Device for generating droplets

ABSTRACT

A system, including method and apparatus, for generating droplets suitable for droplet-based assays. The disclosed systems may include either one-piece or multi-piece droplet generation components configured to form sample-containing droplets by merging aqueous, sample-containing fluid with a background emulsion fluid such as oil, to form an emulsion of sample-containing droplets suspended in the background fluid. In some cases, the disclosed systems may include channels or other suitable mechanisms configured to transport the sample-containing droplets to an outlet region, so that subsequent assay steps may be performed.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/397,774, filed Apr. 29, 2019, now U.S. Pat. No. 10,744,506, which, inturn, is a continuation of U.S. patent application Ser. No. 16/160,783,filed Oct. 15, 2018, now U.S. Pat. No. 10,272,432, which, in turn, is acontinuation of U.S. patent application Ser. No. 15/357,840, filed Nov.21, 2016, now U.S. Pat. No. 10,099,219, which, in turn, is acontinuation of U.S. patent application Ser. No. 13/341,669, filed Dec.30, 2011, now U.S. Pat. No. 9,500,664, which, in turn, is a continuationof PCT Patent Application Serial No. PCT/US2011/030101, filed Mar. 25,2011, which, in turn, is based upon and claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No.61/341,218, filed Mar. 25, 2010. Each of these priority applications isincorporated herein by reference in its entirety for all purposes.

CROSS-REFERENCES TO OTHER MATERIALS

This application incorporates by reference in their entireties for allpurposes the following materials: U.S. Pat. No. 7,041,481, issued May 9,2006; U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010; and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCESPECTROSCOPY (2nd Ed. 1999).

INTRODUCTION

Many biomedical applications rely on high-throughput assays of samplescombined with reagents. For example, in research and clinicalapplications, high-throughput genetic tests using target-specificreagents can provide high-quality information about samples for drugdiscovery, biomarker discovery, and clinical diagnostics, among others.As another example, infectious disease detection often requiresscreening a sample for multiple genetic targets to generatehigh-confidence results.

The trend is toward reduced volumes and detection of more targets.However, creating and mixing smaller volumes can require more complexinstrumentation, which increases cost. Accordingly, improved technologyis needed to permit testing greater numbers of samples and combinationsof samples and reagents, at a higher speed, a lower cost, and/or withreduced instrument complexity.

Emulsions hold substantial promise for revolutionizing high-throughputassays. Emulsification techniques can create billions of aqueousdroplets that function as independent reaction chambers for biochemicalreactions. For example, an aqueous sample (e.g., 200 microliters) can bepartitioned into droplets (e.g., four million droplets of 50 picoliterseach) to allow individual sub-components (e.g., cells, nucleic acids,proteins) to be manipulated, processed, and studied discretely in amassively high-throughput manner.

Splitting a sample into droplets offers numerous advantages. Smallreaction volumes (picoliters to nanoliters) can be utilized, allowingearlier detection by increasing reaction rates and forming moreconcentrated products. Also, a much greater number of independentmeasurements (thousands to millions) can be made on the sample, whencompared to conventional bulk volume reactions performed on a micoliterscale. Thus, the sample can be analyzed more accurately (i.e., morerepetitions of the same test) and in greater depth (i.e., a greaternumber of different tests). In addition, small reaction volumes use lessreagent, thereby lowering the cost per test of consumables. Furthermore,microfluidic technology can provide control over processes used for thegeneration, mixing, incubation, splitting, sorting, and detection ofdroplets, to attain repeatable droplet-based measurements.

Aqueous droplets can be suspended in oil to create a water-in-oilemulsion (W/O). The emulsion can be stabilized with a surfactant toreduce or prevent coalescence of droplets during heating, cooling, andtransport, thereby enabling thermal cycling to be performed.Accordingly, emulsions have been used to perform single-copyamplification of nucleic acid target molecules in droplets using thepolymerase chain reaction (PCR).

Compartmentalization of single molecules of a nucleic acid target indroplets of an emulsion alleviates problems encountered in amplificationof larger sample volumes. In particular, droplets can promote moreefficient and uniform amplification of targets from samples containingcomplex heterogeneous nucleic acid populations, because samplecomplexity in each droplet is reduced. The impact of factors that leadto biasing in bulk amplification, such as amplification efficiency, G+Ccontent, and amplicon annealing, can be minimized by dropletcompartmentalization. Unbiased amplification can be critical indetection of rare species, such as pathogens or cancer cells, thepresence of which could be masked by a high concentration of backgroundspecies in complex clinical samples.

Despite their allure, emulsion-based assays present technical challengesfor high-throughput testing, which can require creation of tens,hundreds, thousands, or even millions of individual samples andsample/reagent combinations. Thus, there is a need for improvedtechniques for the generation, mixing, incubation, splitting, sorting,and detection of droplets.

SUMMARY

The present disclosure provides systems, including methods andapparatus, for generating droplets suitable for droplet-based assays.The disclosed systems may include either one-piece or multi-piecedroplet generation components configured to form sample-containingdroplets by merging aqueous, sample-containing fluid with a backgroundemulsion fluid such as oil, to form an emulsion of sample-containingdroplets suspended in the background fluid. In some cases, the disclosedsystems may include channels or other suitable mechanisms configured totransport the sample-containing droplets to an outlet region, so thatsubsequent assay steps may be performed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of an exemplary droplet generator, in accordancewith aspects of the present disclosure.

FIG. 2 is a top view of another exemplary droplet generator, inaccordance with aspects of the present disclosure.

FIG. 3 is a top view of another exemplary droplet generator, inaccordance with aspects of the present disclosure.

FIG. 4 is a schematic top view of an exemplary droplet generationregion, in accordance with aspects of the present disclosure.

FIG. 5 is a schematic top view of another exemplary droplet generationregion, in accordance with aspects of the present disclosure.

FIG. 6 is a schematic top view of another exemplary droplet generationregion, in accordance with aspects of the present disclosure.

FIG. 7 is an isometric view of four different droplet generators,illustrating the relationship between various cross-type dropletgenerators, in accordance with aspects of the present disclosure.

FIG. 8 is an isometric view of a top surface of a planar-mode dropletgeneration system, in accordance with aspects of the present disclosure.

FIG. 9 is an isometric view of a bottom surface of the dropletgeneration system of FIG. 8.

FIG. 10 is a magnified view of a portion of the bottom surface of thedroplet generation system shown in FIG. 9.

FIG. 11 is a magnified view of an air trap region suitable for use witha planar-mode droplet generation system, in accordance with aspects ofthe present disclosure.

FIG. 12 is a magnified view of another air trap region suitable for usewith a planar-mode droplet generation system, in accordance with aspectsof the present disclosure.

FIG. 13 is a semi-transparent top view of another exemplary dropletgeneration system, in accordance with aspects of the present disclosure.

FIG. 14 is a semi-transparent top view of yet another exemplary dropletgeneration system, in accordance with aspects of the present disclosure.

FIG. 15 is a top view of still another exemplary droplet generationsystem, in accordance with aspects of the present disclosure.

FIG. 16 is a bottom view of the droplet generation system of FIG. 15.

FIG. 17 is a sectional view taken along the line 17-17 in FIG. 16.

FIG. 18 is a top view of yet another exemplary droplet generationsystem, in accordance with aspects of the present disclosure.

FIG. 19 is an isometric view of a magnified portion of the dropletgeneration system of FIG. 18.

FIG. 20 is a top view of still another exemplary droplet generationsystem, in accordance with aspects of the present disclosure.

FIG. 21 is an isometric view of a magnified portion of the dropletgeneration system of FIG. 20.

FIG. 22 is a top view of still another exemplary droplet generationsystem, in accordance with aspects of the present disclosure.

FIG. 23 is an isometric view of a magnified portion of the dropletgeneration system of FIG. 22.

FIG. 24 is a bottom view of a portion of a droplet generation systemaccording to the present teachings, showing channel networks suitablefor use in conjunction with some of the other systems described herein.

FIG. 25 is an isometric view of a droplet generator tube, in accordancewith aspects of the present teachings.

FIG. 26 is an exploded isometric view of another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 27 is an assembled view of the droplet generation system of FIG.26.

FIG. 28 is a partially transparent isometric view of a portion of stillanother droplet generation system, in accordance with aspects of thepresent teachings.

FIG. 29 is a partially transparent isometric view of the portion of thedroplet generation system shown in FIG. 28 assembled with a dropletgeneration housing.

FIG. 30 is an elevational view of still another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 31 is a magnified sectional view of a portion of the dropletgeneration system of FIG. 30.

FIG. 32 is an exploded isometric view of another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 33 is an exploded isometric view of yet another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 34 is an exploded isometric view of a portion of the dropletgeneration system of FIG. 33.

FIG. 35 is an exploded isometric view of still another dropletgeneration system, in accordance with aspects of the present teachings.

FIG. 36 is a magnified sectional view of a portion of the dropletgeneration system of FIG. 35.

FIG. 37 is a magnified sectional view of another portion of the dropletgeneration system of FIG. 35.

FIG. 38 is a magnified top view of another portion of the dropletgeneration system of FIG. 35.

FIG. 39 is a partially transparent isometric view of still anotherdroplet generation system, in accordance with aspects of the presentteachings.

FIG. 40 is a sectional view of the droplet generation system of FIG. 39.

FIG. 41 is a partially transparent isometric view of yet another dropletgeneration system, in accordance with aspects of the present teachings.

FIG. 42 is a sectional view of the droplet generation system of FIG. 41.

FIG. 43 is a partially transparent isometric view of another dropletgeneration system, in accordance with aspects of the present teachings.

FIG. 44 is a magnified sectional view of a portion of the dropletgeneration system of FIG. 43.

FIG. 45 is a partially transparent isometric view of still anotherdroplet generation system, in accordance with aspects of the presentteachings.

FIG. 46 is a magnified sectional view of a portion of the dropletgeneration system of FIG. 45.

FIG. 47 is an exploded isometric view of a disk stack of the dropletgeneration system of FIG. 45.

FIG. 48 is a stylized sectional view of another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 49 is a stylized sectional view of still another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 50 is a stylized sectional view of yet another droplet generationsystem, in accordance with aspects of the present teachings.

FIG. 51 is a flow chart depicting a method of generatingsample-containing droplets, in accordance with aspects of the presentteachings.

FIG. 52 is a flow chart depicting another method of generatingsample-containing droplets, in accordance with aspects of the presentteachings.

DETAILED DESCRIPTION

The present disclosure provides systems, including apparatus andmethods, for generating droplets suitable for droplet-based assays.Droplet generation systems according to the present teachings may bepart of an overall assay system configured to test for the presence ofone or more target molecules in a sample. These overall systems mayinclude methods and apparatus for (A) preparing a sample, such as aclinical or environmental sample, for analysis, (B) separatingcomponents of the samples by partitioning them into droplets or otherpartitions, each containing only about one component (such as a singlecopy of a nucleic acid target or other analyte of interest), (C)amplifying or otherwise reacting the components within the droplets, (D)detecting the amplified or reacted components, or characteristicsthereof, and/or (E) analyzing the resulting data. In this way, complexsamples may be converted into a plurality of simpler, more easilyanalyzed samples, with concomitant reductions in background and assaytimes.

Droplet generation systems according to the present teachings mayinvolve, among others, the following four modes of droplet generation:(A) planar mode droplet generation, (B) continuous mode dropletgeneration, (C) two-part mode droplet generation, and (D) single holemode droplet generation. Droplet generation systems according to eachmode share the characteristic that portions of the system exposed to asample are configured to be disposable, whereas other portions of thesystem may be reusable for multiple different samples. Features of thevarious modes, as well as exemplary embodiments corresponding to eachmode, will be described in detail below, in the following sections: (I)definitions, (II) general principles of droplet generation, (III) planarmode examples, (IV) continuous mode examples, (V) two-part modeexamples, (VI) single hole mode examples, (VII) methods of operation,and (VIII) exemplary numbered paragraphs.

I. Definitions

Technical terms used in this disclosure have the meanings that arecommonly recognized by those skilled in the art. However, the followingterms may have additional meanings, as described below.

Emulsion—a composition comprising liquid droplets disposed in animmiscible carrier fluid, which also is liquid. The carrier fluid, alsotermed a background fluid, forms a continuous phase, which may be termeda carrier phase, a carrier, and/or a background phase. The droplets(e.g., aqueous droplets) are formed by at least one droplet fluid, alsotermed a foreground fluid, which is a liquid and which forms a dropletphase (which may be termed a dispersed phase or discontinuous phase).The droplet phase is immiscible with the continuous phase, which meansthat the droplet phase (i.e., the droplets) and the continuous phase(i.e., the carrier fluid) do not mix to attain homogeneity. The dropletsare isolated from one another by the continuous phase and encapsulated(i.e., enclosed/surrounded) by the continuous phase.

The droplets of an emulsion may have any uniform or non-uniformdistribution in the continuous phase. If non-uniform, the concentrationof the droplets may vary to provide one or more regions of higherdroplet density and one or more regions of lower droplet density in thecontinuous phase. For example, droplets may sink or float in thecontinuous phase, may be clustered in one or more packets along achannel, may be focused toward the center or perimeter of a flow stream,or the like. When droplets are said to be “suspended in the backgroundfluid,” this is intended to cover all of these possibilities.

Any of the emulsions disclosed herein may be monodisperse, that is,composed of droplets of at least generally uniform size, or may bepolydisperse, that is, composed of droplets of various sizes. Ifmonodisperse, the droplets of the emulsion may, for example, vary involume by a standard deviation that is less than about plus or minus100%, 50%, 20%, 10%, 5%, 2%, or 1% of the average droplet volume.Droplets generated from an orifice may be monodisperse or polydisperse.

An emulsion may have any suitable composition. The emulsion may becharacterized by the predominant liquid compound or type of liquidcompound in each phase. The predominant liquid compounds in the emulsionmay be water and oil. “Oil” is any liquid compound or mixture of liquidcompounds that is immiscible with water and that has a high content ofcarbon. In some examples, oil also may have a high content of hydrogen,fluorine, silicon, oxygen, or any combination thereof, among others. Forexample, any of the emulsions disclosed herein may be a water-in-oil(W/O) emulsion (i.e., aqueous droplets in a continuous oil phase). Theoil may, for example, be or include at least one silicone oil, mineraloil, fluorocarbon oil, vegetable oil, or a combination thereof, amongothers. Any other suitable components may be present in any of theemulsion phases, such as at least one surfactant, reagent, sample (i.e.,partitions thereof), other additive, label, particles, or anycombination thereof.

Standard emulsions become unstable when heated (e.g., to temperaturesabove 60° C.) when they are in a packed state (e.g., each droplet isnear a neighboring droplet), because heat generally lowers interfacialtensions, which can lead to droplet coalescence. Thus, standard packedemulsions do not maintain their integrity during high-temperaturereactions, such as PCR, unless emulsion droplets are kept out of contactwith one another or additives (e.g., other oil bases, surfactants, etc.)are used to modify the stability conditions (e.g., interfacial tension,viscosity, steric hindrance, etc.). For example, the droplets may bearranged in single file and spaced from one another along a channel topermit thermal cycling in order to perform PCR. However, following thisapproach using a standard emulsion does not permit a high density ofdroplets, thereby substantially limiting throughput in droplet-basedassays.

Any emulsion disclosed herein may be a heat-stable emulsion. Aheat-stable emulsion is any emulsion that resists coalescence whenheated to at least 50° C. A heat-stable emulsion may be a PCR-stableemulsion, which is an emulsion that resists coalescence throughout thethermal cycling of PCR (e.g., to permit performance of digital PCR).Accordingly, a PCR-stable emulsion may be resistant to coalescence whenheated to at least 80° C. or 90° C., among others. Due to heatstability, a PCR-stable emulsion, in contrast to a standard emulsion,enables PCR assays to be performed in droplets that remain substantiallymonodisperse throughout thermal cycling. Accordingly, digital PCR assayswith PCR-stable emulsions may be substantially more quantitative thanwith standard emulsions. An emulsion may be formulated as PCR stable by,for example, proper selection of carrier fluid and surfactants, amongothers. An exemplary oil formulation to generate PCR-stable emulsionsfor flow-through assays is as follows: (1) Dow Corning 5225C FormulationAid (10% active ingredient in decamethylcyclopentasiloxane)—20% w/w, 2%w/w final concentration active ingredient, (2) Dow Corning 749 Fluid(50% active ingredient in decamethylcyclopentasiloxane)—5% w/w, 2.5% w/wactive ingredient, and (3) Poly(dimethylsiloxane) Dow Corning 200®fluid, viscosity 5.0 cSt (25° C.)—75% w/w. An exemplary oil formulationto generate PCR-stable emulsions for batch assays is as follows: (1) DowCorning 5225C Formulation Aid (10% active ingredient indecamethylcyclopentasiloxane)—20% w/w, 2% w/w final concentration activeingredient, (2) Dow Corning 749 Fluid (50% active ingredient indecamethylcyclopentasiloxane)—60% w/w, 30% w/w active ingredient, and(3) Poly(dimethylsiloxane) Dow Corning 200® fluid, viscosity 5.0 cSt(25° C.)—20% w/w.

Partition—a separated portion of a bulk volume. The partition may be asample partition generated from a sample, such as a prepared sample,that forms the bulk volume. Partitions generated from a bulk volume maybe substantially uniform in size or may have distinct sizes (e.g., setsof partitions of two or more discrete, uniform sizes). Exemplarypartitions are droplets. Partitions may also vary continuously in sizewith a predetermined size distribution or with a random sizedistribution.

Droplet—a small volume of liquid, typically with a spherical shape,encapsulated by an immiscible fluid, such as a continuous phase of anemulsion. The volume of a droplet, and/or the average volume of dropletsin an emulsion, may, for example, be less than about one microliter(i.e., a “microdroplet”) (or between about one microliter and onenanoliter or between about one microliter and one picoliter), less thanabout one nanoliter (or between about one nanoliter and one picoliter),or less than about one picoliter (or between about one picoliter and onefemtoliter), among others. A droplet (or droplets of an emulsion) mayhave a diameter (or an average diameter) of less than about 1000, 100,or 10 micrometers, or of about 1000 to 10 micrometers, among others. Adroplet may be spherical or nonspherical. A droplet may be a simpledroplet or a compound droplet, that is, a droplet in which at least onedroplet encapsulates at least one other droplet.

Surfactant—a surface-active agent capable of reducing the surfacetension of a liquid in which it is dissolved, and/or the interfacialtension with another phase. A surfactant, which also or alternativelymay be described as a detergent and/or a wetting agent, incorporatesboth a hydrophilic portion and a hydrophobic portion, which collectivelyconfer a dual hydrophilic-lipophilic character on the surfactant. Asurfactant may be characterized according to a Hydrophile-LipophileBalance (HLB) value, which is a measure of the surfactant'shydrophilicity compared to its lipophilicity. HLB values range from 0-60and define the relative affinity of a surfactant for water and oil.Nonionic surfactants generally have HLB values ranging from 0-20 andionic surfactants may have HLB values of up to 60. Hydrophilicsurfactants have HLB values greater than about 10 and a greater affinityfor water than oil. Lipophilic surfactants have HLB values less thanabout 10 and a greater affinity for oil than water. The emulsionsdisclosed herein and/or any phase thereof, may include at least onehydrophilic surfactant, at least one lipophilic surfactant, or acombination thereof. Alternatively, or in addition, the emulsionsdisclosed herein and/or any phase thereof, may include at least onenonionic (and/or ionic) detergent. Furthermore, an emulsion disclosedherein and/or any phase thereof may include a surfactant comprisingpolyethyleneglycol, polypropyleneglycol, or Tween 20, among others.

Packet—a set of droplets or other isolated partitions disposed in thesame continuous volume or volume region of a continuous phase. A packetthus may, for example, constitute all of the droplets of an emulsion ormay constitute a segregated fraction of such droplets at a positionalong a channel. Typically, a packet refers to a collection of dropletsthat when analyzed in partial or total give a statistically relevantsampling to quantitatively make a prediction regarding a property of theentire starting sample from which the initial packet of droplets wasmade. The packet of droplets also indicates a spatial proximity betweenthe first and the last droplets of the packet in a channel.

As an analogy with information technology, each droplet serves as a“bit” of information that may contain sequence specific information froma target analyte within a starting sample. A packet of droplets is thenthe sum of all these “bits” of information that together providestatistically relevant information on the analyte of interest from thestarting sample. As with a binary computer, a packet of droplets isanalogous to the contiguous sequence of bits that comprises the smallestunit of binary data on which meaningful computations can be applied. Apacket of droplets can be encoded temporally and/or spatially relativeto other packets that are also disposed in a continuous phase (such asin a flow stream), and/or with the addition of other encoded information(optical, magnetic, etc.) that uniquely identifies the packet relativeto other packets.

Test—a procedure(s) and/or reaction(s) used to characterize a sample,and any signal(s), value(s), data, and/or result(s) obtained from theprocedure(s) and/or reaction(s). A test also may be described as anassay. Exemplary droplet-based assays are biochemical assays usingaqueous assay mixtures. More particularly, the droplet-based assays maybe enzyme assays and/or binding assays, among others. The enzyme assaysmay, for example, determine whether individual droplets contain a copyof a substrate molecule (e.g., a nucleic acid target) for an enzymeand/or a copy of an enzyme molecule. Based on these assay results, aconcentration and/or copy number of the substrate and/or the enzyme in asample may be estimated.

Reaction—a chemical reaction, a binding interaction, a phenotypicchange, or a combination thereof, which generally provides a detectablesignal (e.g., a fluorescence signal) indicating occurrence and/or anextent of occurrence of the reaction. An exemplary reaction is an enzymereaction that involves an enzyme-catalyzed conversion of a substrate toa product.

Any suitable enzyme reactions may be performed in the droplet-basedassays disclosed herein. For example, the reactions may be catalyzed bya kinase, nuclease, nucleotide cyclase, nucleotide ligase, nucleotidephosphodiesterase, polymerase (DNA or RNA), prenyl transferase,pyrophospatase, reporter enzyme (e.g., alkaline phosphatase,beta-galactosidase, chloramphenicol acetyl transferse, glucuronidase,horse radish peroxidase, luciferase, etc.), reverse transcriptase,topoisomerase, etc.

Sample—a compound, composition, and/or mixture of interest, from anysuitable source(s). A sample is the general subject of interest for atest that analyzes an aspect of the sample, such as an aspect related toat least one analyte that may be present in the sample. Samples may beanalyzed in their natural state, as collected, and/or in an alteredstate, for example, following storage, preservation, extraction, lysis,dilution, concentration, purification, filtration, mixing with one ormore reagents, pre-amplification (e.g., to achieve target enrichment byperforming limited cycles (e.g., <15) of PCR on sample prior to PCR),removal of amplicon (e.g., treatment with uracil-d-glycosylase (UDG)prior to PCR to eliminate any carry-over contamination by a previouslygenerated amplicon (i.e., the amplicon is digestable with UDG because itis generated with dUTP instead of dTTP)), partitioning, or anycombination thereof, among others. Clinical samples may includenasopharyngeal wash, blood, plasma, cell-free plasma, buffy coat,saliva, urine, stool, sputum, mucous, wound swab, tissue biopsy, milk, afluid aspirate, a swab (e.g., a nasopharyngeal swab), and/or tissue,among others. Environmental samples may include water, soil, aerosol,and/or air, among others. Research samples may include cultured cells,primary cells, bacteria, spores, viruses, small organisms, any of theclinical samples listed above, or the like. Additional samples mayinclude foodstuffs, weapons components, biodefense samples to be testedfor bio-threat agents, suspected contaminants, and so on.

Samples may be collected for diagnostic purposes (e.g., the quantitativemeasurement of a clinical analyte such as an infectious agent) or formonitoring purposes (e.g., to determine that an environmental analyte ofinterest such as a bio-threat agent has exceeded a predeterminedthreshold).

Analyte—a component(s) or potential component(s) of a sample that isanalyzed in a test. An analyte is a specific subject of interest in atest where the sample is the general subject of interest. An analytemay, for example, be a nucleic acid, protein, peptide, enzyme, cell,bacteria, spore, virus, organelle, macromolecular assembly, drugcandidate, lipid, carbohydrate, metabolite, or any combination thereof,among others. An analyte may be tested for its presence, activity,and/or other characteristic in a sample and/or in partitions thereof.The presence of an analyte may relate to an absolute or relative number,concentration, binary assessment (e.g., present or absent), or the like,of the analyte in a sample or in one or more partitions thereof. In someexamples, a sample may be partitioned such that a copy of the analyte isnot present in all of the partitions, such as being present in thepartitions at an average concentration of about 0.0001 to 10,000, 0.001to 1000, 0.01 to 100, 0.1 to 10, or one copy per partition.

Reagent—a compound, set of compounds, and/or composition that iscombined with a sample in order to perform a particular test(s) on thesample. A reagent may be a target-specific reagent, which is any reagentcomposition that confers specificity for detection of a particulartarget(s) or analyte(s) in a test. A reagent optionally may include achemical reactant and/or a binding partner for the test. A reagent may,for example, include at least one nucleic acid, protein (e.g., anenzyme), cell, virus, organelle, macromolecular assembly, potentialdrug, lipid, carbohydrate, inorganic substance, or any combinationthereof, and may be an aqueous composition, among others. In exemplaryembodiments, the reagent may be an amplification reagent, which mayinclude at least one primer or at least one pair of primers foramplification of a nucleic acid target, at least one probe and/or dye toenable detection of amplification, a polymerase, nucleotides (dNTPsand/or NTPs), divalent magnesium ions, potassium chloride, buffer, orany combination thereof, among others.

Nucleic acid—a compound comprising a chain of nucleotide monomers. Anucleic acid may be single-stranded or double-stranded (i.e.,base-paired with another nucleic acid), among others. The chain of anucleic acid may be composed of any suitable number of monomers, such asat least about ten or one-hundred, among others. Generally, the lengthof a nucleic acid chain corresponds to its source, with syntheticnucleic acids (e.g., primers and probes) typically being shorter, andbiologically/enzymatically generated nucleic acids (e.g., nucleic acidanalytes) typically being longer.

A nucleic acid may have a natural or artificial structure, or acombination thereof. Nucleic acids with a natural structure, namely,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally have abackbone of alternating pentose sugar groups and phosphate groups. Eachpentose group is linked to a nucleobase (e.g., a purine (such as adenine(A) or guanine (T)) or a pyrimidine (such as cytosine (C), thymine (T),or uracil (U))). Nucleic acids with an artificial structure are analogsof natural nucleic acids and may, for example, be created by changes tothe pentose and/or phosphate groups of the natural backbone. Exemplaryartificial nucleic acids include glycol nucleic acids (GNA), peptidenucleic acids (PNA), locked nucleic acid (LNA), threose nucleic acids(TNA), and the like.

The sequence of a nucleic acid is defined by the order in whichnucleobases are arranged along the backbone. This sequence generallydetermines the ability of the nucleic acid to bind specifically to apartner chain (or to form an intramolecular duplex) by hydrogen bonding.In particular, adenine pairs with thymine (or uracil) and guanine pairswith cytosine. A nucleic acid that can bind to another nucleic acid inan antiparallel fashion by forming a consecutive string of such basepairs with the other nucleic acid is termed “complementary.”

Replication—a process forming a copy (i.e., a direct copy and/or acomplementary copy) of a nucleic acid or a segment thereof. Replicationgenerally involves an enzyme, such as a polymerase and/or a ligase,among others. The nucleic acid and/or segment replicated is a template(and/or a target) for replication.

Amplification—a reaction in which replication occurs repeatedly overtime to form multiple copies of at least one segment of a templatemolecule. Amplification may generate an exponential or linear increasein the number of copies as amplification proceeds. Typicalamplifications produce a greater than 1,000-fold increase in copy numberand/or signal. Exemplary amplification reactions for the droplet-basedassays disclosed herein may include the polymerase chain reaction (PCR)or ligase chain reaction, each of which is driven by thermal cycling.The droplet-based assays also or alternatively may use otheramplification reactions, which may be performed isothermally, such asbranched-probe DNA assays, cascade-RCA, helicase-dependentamplification, loop-mediated isothermal amplification (LAMP), nucleicacid based amplification (NASBA), nicking enzyme amplification reaction(NEAR), PAN-AC, Q-beta replicase amplification, rolling circlereplication (RCA), self-sustaining sequence replication,strand-displacement amplification, and the like. Amplification mayutilize a linear or circular template.

Amplification may be performed with any suitable reagents. Amplificationmay be performed, or tested for its occurrence, in an amplificationmixture, which is any composition capable of generating multiple copiesof a nucleic acid target molecule, if present, in the composition. Anamplification mixture may include any combination of at least one primeror primer pair, at least one probe, at least one replication enzyme(e.g., at least one polymerase, such as at least one DNA and/or RNApolymerase), and deoxynucleotide (and/or nucleotide) triphosphates(dNTPs and/or NTPs), among others. Further aspects of assay mixtures anddetection strategies that enable multiplexed amplification and detectionof two or more target species in the same droplet are describedelsewhere herein, such as in Section X, among others.

PCR—nucleic acid amplification that relies on alternating cycles ofheating and cooling (i.e., thermal cycling) to achieve successive roundsof replication. PCR may be performed by thermal cycling between two ormore temperature set points, such as a higher melting (denaturation)temperature and a lower annealing/extension temperature, or among threeor more temperature set points, such as a higher melting temperature, alower annealing temperature, and an intermediate extension temperature,among others. PCR may be performed with a thermostable polymerase, suchas Taq DNA polymerase (e.g., wild-type enzyme, a Stoffel fragment,FastStart polymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tthpolymerase, Vent polymerase, or a combination thereof, among others. PCRgenerally produces an exponential increase in the amount of a productamplicon over successive cycles.

Any suitable PCR methodology or combination of methodologies may beutilized in the droplet-based assays disclosed herein, such asallele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, endpointPCR, hot-start PCR, in situ PCR, intersequence-specific PCR, inversePCR, linear after exponential PCR, ligation-mediated PCR,methylation-specific PCR, miniprimer PCR, multiplex ligation-dependentprobe amplification, multiplex PCR, nested PCR, overlap-extension PCR,polymerase cycling assembly, qualitative PCR, quantitative PCR,real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermalasymmetric interlaced PCR, touchdown PCR, or universal fast walking PCR,among others.

Digital PCR—PCR performed on portions of a sample to determine thepresence/absence, concentration, and/or copy number of a nucleic acidtarget in the sample, based on how many of the sample portions supportamplification of the target. Digital PCR may (or may not) be performedas endpoint PCR. Digital PCR may (or may not) be performed as real-timePCR for each of the partitions.

PCR theoretically results in an exponential amplification of a nucleicacid sequence (analyte) from a sample. By measuring the number ofamplification cycles required to achieve a threshold level ofamplification (as in real-time PCR), one can theoretically calculate thestarting concentration of nucleic acid. In practice, however, there aremany factors that make the PCR process non-exponential, such as varyingamplification efficiencies, low copy numbers of starting nucleic acid,and competition with background contaminant nucleic acid. Digital PCR isgenerally insensitive to these factors, since it does not rely on theassumption that the PCR process is exponential. In digital PCR,individual nucleic acid molecules are separated from the initial sampleinto partitions, then amplified to detectable levels. Each partitionthen provides digital information on the presence or absence of eachindividual nucleic acid molecule within each partition. When enoughpartitions are measured using this technique, the digital informationcan be consolidated to make a statistically relevant measure of startingconcentration for the nucleic acid target (analyte) in the sample.

The concept of digital PCR may be extended to other types of analytes,besides nucleic acids. In particular, a signal amplification reactionmay be utilized to permit detection of a single copy of a molecule ofthe analyte in individual droplets, to permit data analysis of dropletsignals for other analytes in the manner described in Section VII (e.g.,using an algorithm based on Poisson statistics). Exemplary signalamplification reactions that permit detection of single copies of othertypes of analytes in droplets include enzyme reactions.

Qualitative PCR—a PCR-based analysis that determines whether or not atarget is present in a sample, generally without any substantialquantification of target presence. In exemplary embodiments, digital PCRthat is qualitative may be performed by determining whether a packet ofdroplets contains at least a predefined percentage of positive droplets(a positive sample) or not (a negative sample).

Quantitative PCR—a PCR-based analysis that determines a concentrationand/or copy number of a target in a sample.

RT-PCR (reverse transcription-PCR)—PCR utilizing a complementary DNAtemplate produced by reverse transcription of RNA. RT-PCR permitsanalysis of an RNA sample by (1) forming complementary DNA copies ofRNA, such as with a reverse transcriptase enzyme, and (2) PCRamplification using the complementary DNA as a template. In someembodiments, the same enzyme, such as Tth polymerase, may be used forreverse transcription and PCR.

Real-time PCR—a PCR-based analysis in which amplicon formation ismeasured during the reaction, such as after completion of one or morethermal cycles prior to the final thermal cycle of the reaction.Real-time PCR generally provides quantification of a target based on thekinetics of target amplification.

Endpoint PCR—a PCR-based analysis in which amplicon formation ismeasured after the completion of thermal cycling.

Amplicon—a product of an amplification reaction. An amplicon may besingle-stranded or double-stranded, or a combination thereof. Anamplicon corresponds to any suitable segment or the entire length of anucleic acid target.

Primer—a nucleic acid capable of, and/or used for, priming replicationof a nucleic acid template. Thus, a primer is a shorter nucleic acidthat is complementary to a longer template. During replication, theprimer is extended, based on the template sequence, to produce a longernucleic acid that is a complementary copy of the template. A primer maybe DNA, RNA, an analog thereof (i.e., an artificial nucleic acid), orany combination thereof. A primer may have any suitable length, such asat least about 10, 15, 20, or 30 nucleotides. Exemplary primers aresynthesized chemically. Primers may be supplied as at least one pair ofprimers for amplification of at least one nucleic acid target. A pair ofprimers may be a sense primer and an antisense primer that collectivelydefine the opposing ends (and thus the length) of a resulting amplicon.

Probe—a nucleic acid connected to at least one label, such as at leastone dye. A probe may be a sequence-specific binding partner for anucleic acid target and/or amplicon. The probe may be designed to enabledetection of target amplification based on fluorescence resonance energytransfer (FRET). An exemplary probe for the nucleic acid assaysdisclosed herein includes one or more nucleic acids connected to a pairof dyes that collectively exhibit fluorescence resonance energy transfer(FRET) when proximate one another. The pair of dyes may provide firstand second emitters, or an emitter and a quencher, among others.Fluorescence emission from the pair of dyes changes when the dyes areseparated from one another, such as by cleavage of the probe duringprimer extension (e.g., a 5′ nuclease assay, such as with a TAQMANprobe), or when the probe hybridizes to an amplicon (e.g., a molecularbeacon probe).

The nucleic acid portion of the probe may have any suitable structure ororigin, for example, the portion may be a locked nucleic acid, a memberof a universal probe library, or the like. In other cases, a probe andone of the primers of a primer pair may be combined in the same molecule(e.g., AMPLIFLUOR primers or SCORPION primers). As an example, theprimer-probe molecule may include a primer sequence at its 3′ end and amolecular beacon-style probe at its 5′ end. With this arrangement,related primer-probe molecules labeled with different dyes can be usedin a multiplexed assay with the same reverse primer to quantify targetsequences differing by a single nucleotide (single nucleotidepolymorphisms (SNPs)). Another exemplary probe for droplet-based nucleicacid assays is a Plexor primer.

Label—an identifying and/or distinguishing marker or identifierconnected to or incorporated into any entity, such as a compound,biological particle (e.g., a cell, bacteria, spore, virus, ororganelle), or droplet. A label may, for example, be a dye that rendersan entity optically detectable and/or optically distinguishable.Exemplary dyes used for labeling are fluorescent dyes (fluorophores) andfluorescence quenchers.

Reporter—a compound or set of compounds that reports a condition, suchas the extent of a reaction. Exemplary reporters comprise at least onedye, such as a fluorescent dye or an energy transfer pair, and/or atleast one oligonucleotide. Exemplary reporters for nucleic acidamplification assays may include a probe and/or an intercalating dye(e.g., SYBR Green, ethidium bromide, etc.).

Code—a mechanism for differentiating distinct members of a set.Exemplary codes to differentiate different types of droplets may includedifferent droplet sizes, dyes, combinations of dyes, amounts of one ormore dyes, enclosed code particles, or any combination thereof, amongothers. A code may, for example, be used to distinguish differentpackets of droplets, or different types of droplets within a packet,among others.

Binding partner—a member of a pair of members that bind to one another.Each member may be a compound or biological particle (e.g., a cell,bacteria, spore, virus, organelle, or the like), among others. Bindingpartners may bind specifically to one another. Specific binding may becharacterized by a dissociation constant of less than about 10⁻⁴, 10⁻⁶,10⁻⁸, or 10⁻¹⁶ M. Exemplary specific binding partners include biotin andavidin/streptavidin, a sense nucleic acid and a complementary antisensenucleic acid (e.g., a probe and an amplicon), a primer and its target,an antibody and a corresponding antigen, a receptor and its ligand, andthe like.

Channel—a passage for fluid travel. A channel generally includes atleast one inlet, where fluid enters the channel, and at least oneoutlet, where fluid exits the channel. The functions of the inlet andthe outlet may be interchangeable, that is, fluid may flow through achannel in only one direction or in opposing directions, generally atdifferent times. A channel may include walls that define and enclose thepassage between the inlet and the outlet. A channel may, for example, beformed by a tube (e.g., a capillary tube), in or on a planar structure(e.g., a chip), or a combination thereof, among others. A channel may ormay not branch. A channel may be linear or nonlinear. Exemplarynonlinear channels include a channel extending along a planar flow path(e.g., a serpentine channel) a nonplanar flow path (e.g., a helicalchannel to provide a helical flow path). Any of the channels disclosedherein may be a microfluidic channel, which is a channel having acharacteristic transverse dimension (e.g., the channel's averagediameter) of less than about one millimeter. Channels also may includeone or more venting mechanisms to allow fluid to enter/exit without theneed for an open outlet. Examples of venting mechanisms include but arenot limited to hydrophobic vent openings or the use of porous materialsto either make up a portion of the channel or to block an outlet ifpresent. A channel may or may not be elongate. For example, an elongatechannel may take the form of a four-walled conduit, and a non-elongatechannel may take the form of radial flow between two parallel disks. Forexample, the oil flow in a butted tube droplet generator may flowradially inward in a channel defined by the disk-shaped faces of thebutted tubes.

Fluidics Network—an assembly for manipulating fluid, generally bytransferring fluid between compartments of the assembly and/or bydriving flow of fluid along and/or through one or more flow pathsdefined by the assembly. A fluidics network may include any suitablestructure, such as one or more channels, chambers, reservoirs, valves,pumps, thermal control devices (e.g., heaters/coolers), sensors (e.g.,for measuring temperature, pressure, flow, etc.), or any combinationthereof, among others.

II. General Principles of Droplet Generation

It may be desirable, in systems such as DNA amplification systems, amongothers, to generate sample-containing droplets using a partially orcompletely disposable apparatus. This may be accomplished by adisposable cartridge configured to generate droplets as part of a seriesof sample preparation steps that also may include lysing, purification,and concentration, among others. However, in other cases, it may bedesirable to provide a partially or completely disposable apparatusconfigured to perform droplet generation without performing substantialadditional sample preparation steps. This may be desirable, for example,when the DNA amplification system is configured to analyze samples thatare typically prepared at another location or by a practitioner. Underthese circumstances, a dedicated droplet generation system may be thesimplest and most economical solution.

The components of droplet generation systems described herein mayinclude, for example, substrates, wells (i.e. reservoirs), channels,tubes, and the like. These components may be manufactured by anysuitable method(s) known in the art, for example by injection molding,machining, and/or the like. In some cases, all of the components of adroplet generation system disclosed according to the present teachingsmay be proprietary. In other cases, one or more components of adisclosed system may be available as an off-the-shelf component, whichmay be integrated with other components either with or withoutmodification.

Many configurations of droplet generators may be suitable as componentsof a droplet generation system according to the present teachings. Forexample, suitable droplet generators include butted tubes, tubes drilledwith intersecting channels, tubes partially or completely insertedinside other tubes, and tubes having multiple apertures, among others,where “tubes” means elongate hollow structures of any cross-sectionalshape. Suitable fluid reservoirs include pipette tips, spin columns,wells (either individual or in a plate array), tubes, and syringes,among others. This section describes some general principles of dropletgeneration that apply to the present teachings, and provides a fewspecific examples of droplet generators embodying those principles; seeFIGS. 1-7.

In general, droplets generated according to the present teachings willbe sample-containing droplets suspended in a background fluid such asoil. Droplets of this type may be referred to as “water-in-oil”droplets. “Sample-containing” means that the aqueous fluid from whichthe droplets are formed contains sample material to be analyzed for thepresence of one or more target molecules. The droplets may containadditional components other than sample material. For example, dropletgeneration may be performed after the sample has been modified by mixingit with one or more reagents to form a bulk assay mixture.

Droplet generation may divide the sample fluid or the bulk assay mixtureinto a plurality of partitioned mixtures (and thus sample partitions)that are isolated from one another in respective droplets by anintervening, immiscible carrier fluid. The droplets may be generatedfrom a sample serially, such as from one orifice and/or one dropletgenerator (which may be termed an emulsion generator). Alternatively,the droplets may be generated in parallel from a sample, such as fromtwo or more orifices and/or two or more droplet generators in fluidcommunication with (and/or supplied by) the same sample. As anotherexample, droplets may be generated in parallel from a perforated platedefining an array of orifices. In some examples, the droplets may begenerated in bulk, such as by agitation or sonication, among others. Insome examples, a plurality of emulsions may be generated, eitherserially or in parallel, from a plurality of samples.

Various exemplary droplet generation configurations may be suitable forgenerating water-in-oil droplets containing a mixture of sample andreagent. The generated droplets then may be transported to athermocycling instrument for PCR amplification. Each depictedconfiguration is compatible with continuous production of emulsions andwith any suitable method of pumping, including at leastpressure-controlled pumping, vacuum-controlled pumping, centrifugation,gravity-driven flow, and positive displacement pumping. A dropletgenerator or droplet generation configuration according to the presentdisclosure may be connected to a pressure/pump source located on acomplementary PCR instrument, or may include any pumps and/or pressuresources needed to facilitate droplet generation.

Each depicted droplet configuration in FIGS. 1-6 may be capable ofhigh-throughput droplet generation (1,000 droplets per second) in adisposable device, such as a cartridge. Each configuration may beconstructed in a number of different ways. For example, fluid channelsmay be formed in a single injection molded piece of material, which isthen sealed with a sealing member such as a featureless film or othermaterial layer. Alternatively, fluid channels may be formed by injectionmolding two layers of material that fit together to form the channels,such as cylindrical channels formed by complementary hemisphericalgrooves. The fluid channels of the droplet generation configurationsdepicted in FIGS. 1-6 may have varying channel depths, such as 50, 100,150, 200, or 250 μm, among others. Furthermore, the principles ofdroplet generation that apply to the exemplary droplet generators ofFIGS. 1-6 apply to many droplet generation configurations other thancartridge-based configurations. Several of these alternateconfigurations are described in this disclosure.

FIG. 1 depicts a 3-port cross droplet generation configuration 100wherein oil from a first fluid well (or chamber) 102 is transferredthrough two similar branches of a fluid channel section 104. The oilfrom well 102 intersects with aqueous fluid from a second fluid chamber106, which is transferred along a fluid channel section 108 to anintersection area generally indicated at 110. The oil from well 102arrives at intersection 110 from two different and substantiallyopposite directions, whereas the aqueous solution arrives at theintersection along only a single path that is substantiallyperpendicular to both directions of travel of the arriving oil. Theresult is that at intersection 110, aqueous droplets in an oilbackground (i.e., a water-in-oil emulsion) are produced and transferredalong a fluid channel section 112 to a third chamber 114, where theemulsion can be temporarily stored and/or transferred to a thermocyclinginstrument.

FIG. 2 depicts a configuration 115 that is similar in most respects todroplet generation configuration 100 depicted in FIG. 1. Specifically,in droplet generation configuration 115, oil from a first fluid chamber116 is transferred through two similar branches of a fluid channelsection 118. Fluid channel sections 118 intersect with a fluid channelsection 122 that transfers aqueous fluid from a second fluid chamber120, at an intersection area generally indicated at 124. As inconfiguration 100, the oil from chamber 116 arrives at intersection 110from two different directions, but unlike in configuration 100, the oildoes not arrive from substantially opposite (antiparallel) directions.Rather, channel sections 118 each intersect channel section 122 at anon-perpendicular angle, which is depicted as approximately 60 degreesin FIG. 48B. In general, configuration 115 may include oil fluidchannels that intersect an aqueous fluid channel at any desired angle orangles. Oil flowing through channel sections 118 and aqueous solutionflowing through channel section 122 combine to form a water-in-oilemulsion of aqueous droplets suspended in an oil background. As in thecase of configuration 100, the droplets then may be transferred along afluid channel section 126 to a third fluid chamber 128, for storageand/or transfer to a thermocycling instrument.

FIG. 3 depicts a four-port droplet generation configuration 129 thatincludes two separate oil wells or chambers. A first oil chamber 130 isconfigured to store oil and transfer the oil through a fluid channelsection 132 toward a channel intersection point generally indicated at142. A second oil chamber 134 is similarly configured to store andtransfer oil toward the intersection point through a fluid channelsection 136. An aqueous fluid chamber 138 is configured to store aqueousfluid, such as a sample/reagent mixture, and to transfer the aqueousfluid through fluid channel section 140 toward intersection point 142.When the oil traveling through fluid channel sections 132 and 136intersects with the aqueous fluid traveling through fluid channelsection 140, a water-in-oil emulsion of aqueous droplets suspended inoil is generated. Although fluid channel 140 is depicted as intersectingwith each of fluid channels 132 and 136 at a perpendicular angle, ingeneral the channels may intersect at any desired angle, as describedpreviously with respect to droplet generation configuration 115 of FIG.2. The emulsion generated at intersection 142 travels through outgoingfluid channel section 144 toward an emulsion chamber 146, where theemulsion may be temporarily held for transfer to an instrument, such asa thermocycling instrument.

FIGS. 4-6 schematically depict fluid channel intersection regions ofseveral other possible droplet generation configurations, in which thearrows within the depicted fluid channels indicate the direction offluid flow within each channel. Although fluid chambers for receivingand/or storing oil, water, and any generated emulsion are not depictedin FIGS. 4-6, these chambers or at least some source of oil and aqueousfluid would be present in a cartridge containing any of the depictedconfigurations. The fluid channels and any associated chambers may beformed by any suitable method, such as injection molding complementarysections of thermoplastic as described previously.

FIG. 4 depicts a “single T” configuration 150 in which oil traveling inan oil channel 152 intersects with aqueous fluid traveling in an aqueouschannel 154 at fluid channel intersection 156, to produce a water-in-oilemulsion that travels through outgoing fluid channel 158. Thisconfiguration differs from those of FIGS. 1-3 in that oil arrives at theoil/water intersection from only a single direction. Accordingly,droplets may be formed by a slightly different physical mechanism thanin configurations where oil arrives from two directions. For example,droplets formed in the single T configuration of FIG. 4 may be formedprimarily by a shear mechanism rather than primarily by a compressionmechanism. However, the physics of droplet formation is not completelyunderstood and likely depends on many factors, including the channeldiameters, fluid velocities, and fluid viscosities.

FIG. 5 depicts a “double T” configuration 160 in which oil traveling inan oil channel 162 intersects with aqueous fluid traveling in a firstaqueous channel 164 at a first intersection 166, to produce awater-in-oil emulsion that travels through intermediate fluid channel168. Channel 168 intersects with a second aqueous channel 170 at asecond intersection 172, to generate additional water-in-oil dropletswithin the emulsion. This geometry also may be used to generate doubleemulsions of water-in-oil-in-water droplets, and/or to generate twopopulations of droplets with different compositions. In any case, all ofthe generated droplets then travel through outgoing fluid channel 174.This configuration again differs from those of FIGS. 1-3 in that oilarrives at the oil/water intersections from only a single direction. Inaddition, configuration 160 differs from single T configuration 150depicted in FIG. 4 due to the presence of two oil/water intersections.This may result in a greater density of droplets in the water-in-oilemulsion generated by configuration 160 than in the emulsion generationby configuration 150, which includes only one oil/water intersection.

FIG. 6 depicts a droplet generation configuration 180 in which oiltraveling in an oil channel 182 intersects with aqueous fluid travelingin first and second aqueous channels 184 and 186 at an intersection 188.In this configuration, the aqueous fluid arrives at the intersectionfrom two opposite directions, both of which are substantiallyperpendicular to the direction of travel of the oil in channel 182. Moregenerally, the aqueous fluid can intersect with the oil at any desiredangles. Depending on at least the sizes of the various channels, theflow rates of the oil and the aqueous fluid, and the angle ofintersection of the aqueous fluid channels with the oil channel, aconfiguration of this type may be suitable for producing either anoil-in-water emulsion or a water-in-oil emulsion. In either case, theemulsion will travel away from intersection 188 through outgoing fluidchannel 190.

FIG. 7 illustrates various continuous droplet generators, which arecharacterized by being formed from a single piece of material, and therelationships between them. More specifically, FIG. 7 shows a firstcontinuous droplet generator 200 including a single transverse channelintersecting an inner axial channel, a second continuous dropletgenerator 240 including two transverse channels intersecting an inneraxial channel, a third continuous droplet generator 260 including threetransverse channels intersecting an inner axial channel, and a buttedtube droplet generator 280, which as described below would not typicallybe characterized as a continuous droplet generator. Other continuousdroplet generators similar to these examples are possible, such asgenerators with more than three transverse channels intersecting aninner axial channel, or partially butted type generators in which thetubes remain connected to each other along a portion of theircross-sections.

Droplet generator 200 includes hollow channels 202, 204 that intersectat an intersection region 206. To generate droplets, one of thesechannels will generally carry a foreground fluid toward intersectionregion 206 from one direction, while the other channel carries abackground fluid toward intersection region 206 from both directions.Typically, channel 202 will carry a foreground fluid such as asample-containing solution, and channel 204 will carry a backgroundfluid such as oil, but the opposite is also possible. In any case, anemulsion will be created at intersection region 206 and will continuemoving through channel 202 in the direction of travel of the foregroundfluid, as described in detail above.

Droplet generator 240 includes three hollow channels 242, 244, and 246that intersect at an intersection region 248. To generate droplets,channel 242 will typically carry a foreground fluid such as asample-containing solution toward intersection region 248 from a singledirection, and each of channels 244, 246 will typically carry abackground fluid such as oil toward intersection region 248 from twoopposite directions. In that case, an emulsion will be created atintersection region 248 and will continue moving through channel 242 inthe direction of travel of the foreground fluid. It is also possiblethat each of channels 244, 246 would carry a foreground fluid towardintersection region 248 from a single direction, and channel 242 wouldcarry a background fluid toward intersection region 248 from twoopposite directions. In that case, the emulsion created at intersectionregion 248 would travel through both channels 244 and 246, in theoriginal directions of travel of the foreground fluid in each of thosechannels. Droplet generator 240 thus may function to produce dropletsthat emerge from two separate channels.

Similarly, droplet generator 260 includes four channels 262, 264, 266,268 that intersect to generate an emulsion of foreground fluid dropletsin background fluid at an intersection region 250. By analogy to thethree-channel configuration of droplet generator 240, the four-channelconfiguration of droplet generator 260 may be used either to generate asingle emulsion that travels through channel 262, or to generatedifferent emulsions that travel through channels 264, 266, and 268.

Droplet generator 280 is a butted tube generator that includes a firstsection of hollow tube 282 and a second section of hollow tube 284. Tubesection 282 includes a fluid channel 286, and tube section 284 includesa fluid channel 288. The tube sections are separated by a smalldistance, forming an intersection region 290 between the tubes.Accordingly, if a foreground fluid flows toward intersection region 290through channel 286, and a background fluid flows radially inward towardintersection region 290 from the region outside the tubes, an emulsioncan be created and flow into channel 288.

The progression from droplet generator 200 through droplet generator 280illustrates the relationship between these various droplet generators.Specifically, if the variable n is chosen to represent the number ofradial fluid channels that intersect a longitudinal fluid channel at anintersection region within a tube, then droplet generator 200 may becharacterized as an “n=1” cross-type droplet generator, dropletgenerator 240 may be characterized as an “n=2” cross-type dropletgenerator, droplet generator 260 may be characterized as an “n=3”cross-type droplet generator, and droplet generator 280 may becharacterized as an “n=∞” cross-type droplet generator, because the gapbetween tubes 282 and 284 may be viewed as formed from an infinitenumber of radial fluid channels extending continuously around thecircumference of a single elongate tube. Because droplet generator 280is formed from two separate pieces of material, it would not typicallybe characterized as a continuous or continuous mode droplet generator.

III. Planar Mode Examples

This section describes examples of “planar mode” droplet generators, inwhich sample-containing droplets suspended in a background fluid aregenerated and transported substantially within a plane; see FIGS. 8-24.As used herein, “substantially within a plane” or “substantially planar”means that the radius of curvature of the space in which droplets aregenerated and transported is much greater than the cross-sectionaldimensions of the channels through which the droplets are created andtransported, and the curvature does not substantially alter thehydraulic function of the channels.

In some cases (see, e.g., FIGS. 8-17), well protrusions forsample-containing fluid, background fluid, and droplets may beintegrally formed with a substantially planar substrate of the dropletgenerator. In other cases (see, e.g., FIGS. 18-24), the wells may beformed as one or more separate components, and configured to form asubstantially fluid tight seal or interface with a substantially planarsubstrate of the droplet generator. In intermediate cases, some wellsmay be integrally formed with the substrate, and some may be formed asone or more separate components. Although the Figures focus on the caseswhere the wells are either entirely integrally formed with, or entirelyseparately formed from, the planar substrate, the intermediatepossibilities are also contemplated by the present teachings.

FIG. 8 is a perspective view of a top surface of a planar-mode dropletgenerator, generally indicated at 300, in accordance with aspects of thepresent disclosure. FIG. 9 is a perspective view of a bottom surface ofdroplet generator 300 of FIG. 8. Droplet generator 300 includes asubstantially planar substrate 302 having a top surface 304 and a bottomsurface 306. In the embodiment of FIG. 8, a sample well 308, abackground fluid well 310, and a droplet outlet region (which in thisexample takes the form of a droplet well 312) are integrally formed withsubstrate 302. A network of channels, generally indicated at 314, isformed in the bottom surface 306 of substrate 302 and fluidicallyinterconnects the sample well, the background fluid well, and thedroplet outlet region. In droplet generator 300, eight identical sets ofwells and channels are shown. More generally, any desired number ofwells and channels may be formed with substrate 302. The same principleholds true for all of the planar mode droplet generators described inSection III.

A sealing member 316 (shown in FIG. 8) is configured to be disposedadjacent to the bottom surface of substrate 302, to form a substantiallyfluid tight seal with the bottom surface of the substrate and thus withchannel network 314. Although sealing member 316 is shown in FIG. 8 as afeatureless, substantially planar member, in some cases the network ofchannels may be partially or entirely formed in the sealing memberrather than exclusively in substrate 302. Regardless of whether thechannel network is formed exclusively in the substrate, exclusively inthe sealing member, or partially in each of those components, a fluidtight network of channels will be formed when the substrate and thesealing member are brought together. Furthermore, the sealing member canbe a deformable film that can take on non-planar configurations when itis not bonded to the substrate.

As described in more detail below, a source of pressure will generallybe applied at least to sample well 308 and background fluid well 310,and possibly also to droplet well 312, in order to generate dropletswith droplet generator 300. Accordingly, wells 308, 310, and 312 shouldbe configured to withstand the side forces expected when pressure isapplied, as well as other expected forces such as the forces ofintegration with a pumping unit and the forces expected during shippingand handling. Wells 308, 310, and 312 therefore may have walls that areapproximately 0.20 inches thick. More generally the well walls may havethicknesses in the approximate range from 0.04 to 0.40 inches thick,depending on the expected forces and the material from which dropletgenerator 300 is constructed.

FIG. 10 is a magnified view of a portion of bottom surface 306 ofsubstrate 302, showing further details of channel network 314. Channelnetwork 314 defines a droplet generation region indicated at 320, whichis configured to generate sample-containing droplets suspended in thebackground fluid. More specifically, droplet generation region 320 isdefined by the intersection of a first channel 322, a second channel324, and a third channel 326. First channel 322 is configured totransport sample-containing fluid from sample well 308 to dropletgeneration region 320, second channel 324 is configured to transportbackground fluid from background fluid well 310 to droplet generationregion 320, and third channel 326 is configured to transportsample-containing droplets from droplet generation region 320 to dropletwell 312. Droplets are formed at droplet generation region 320 accordingto principles that have already been described; see, e.g., FIG. 1 andaccompanying discussion above.

Channel network 314 includes various features that can be selected orchanged to affect the droplet generation accomplished by dropletgenerator 300. For example, second channel 324, which transportsbackground fluid from background fluid well 310 to droplet generationregion 320, may (as depicted in FIGS. 9-10) include two background fluidsub-channels 324 a, 324 b, which intersect first channel 322 from twodifferent directions. As a result of the intersection of sub-channels324 a, 324 b with first channel 322 and third channel 326, dropletgeneration region 320 is formed as a cross-shaped intersection region.

When two background fluid sub-channels are used, the two sub-channelsmay be configured to have substantially equal hydraulic resistances, sothat the rate of background fluid flow through each sub-channel issubstantially the same. This may be accomplished, for example, by givingthe sub-channels approximately equal lengths, or by adjusting otherparameters of the sub-channels such as their diameters and/or innersurface characteristics. Furthermore, the two sub-channels may includeenlarged portions 328 a, 328 b in a portion of each sub-channel adjacentto the droplet generation region. These enlarged channel portions may,for example, affect the size of droplets that are generated. Moregenerally, the sizes of the channels remote from the cross can be madebigger or smaller to control the resistance to flow in each channel, andthus the flow rate. The two oil channels are sized (width, depth,length) to give the same resistance so that their flow rates aresubstantially equal. The relative sizes of the oil and sample channelsare selected to give a desired sample to oil flow rate.

As FIG. 10 depicts, channel network 314 also includes an air trap 330disposed along first channel 322, between sample well 308 and dropletgeneration region 320. Air trap 330, which can take various forms, isgenerally configured to prevent sample-containing fluid from beinginadvertently drawn through first channel 322 by capillary action orother forces. Essentially, air trap 330 functions as a simple valve, tostop the flow of sample-containing fluid through first channel 322 untila desired time. This feature may be desirable to avoid uncontrolledemulsion formation.

More generally, air traps according to the present teachings function bypinning a liquid/air interface at a location where the channelcross-section abruptly increases in one or more dimensions. This has theeffect of locally increasing the effective contact angle of theliquid/channel wall interface to a value greater than 90 degrees, whichresults in a local force that stops further liquid movement. Theoperation of the device therefore consists of loading sample into a drydevice before the oil is loaded. The sample flows through its channel(by gravity plus capillarity) to the air trap, where the flow stops dueto the channel expansion at that point. Oil is then loaded and flowsthrough its channels (by gravity plus capillarity) to the cross. Onceoil reaches the cross, any air remaining in the air trap (and thechannel between the air trap and cross) is trapped between the sampleand oil and prevents the two fluids from prematurely coming intocontact. Some oil can flow toward the air trap, being drawn along thecorners of the channel by capillary forces; it bypasses the trapped air.The contraction/expansion features in the air trap slow the advance ofthis oil because capillary forces are reduced when the channeldimensions are expanding. The final result is that the air trap keepsthe sample and oil substantially separated until a fluidic driving forceis applied. This feature is desirable to avoid the uncontrolled emulsionformation that would occur if the oil and sample were allowed to mixprematurely.

FIGS. 11-12 depict exemplary air trap embodiments that may be suitablefor use with a droplet generation system such as droplet generator 300,in accordance with aspects of the present teachings. More specifically,FIG. 11 shows portions of a channel network 314′, including a firstchannel 322′, sub-channels 324 a′, 324 b′ of a second channel 324′, athird channel 326′, and an exemplary air trap 330′ disposed alongchannel 322′. In the example depicted in FIG. 11, components representedby primed reference numbers are configured to perform functions similarto the objects represented by corresponding unprimed reference numbersin FIG. 10. Air trap 330′ includes three sections 330 a′, 330 b′, 330c′, each of which includes at least one bent angle 332 a′, 332 b′, 332c′ around which sample-containing fluid must pass in order to passentirely through air trap 330′. These locations where the channelchanges in width and in depth create additional sites for stopping fluidflow by the capillary pinning mechanism described above.

FIG. 12 shows portions of a channel network 314″, including a firstchannel 322″, sub-channels 324 a″, 324 b″ of a second channel 324″, athird channel 326″, and an exemplary air trap 330″ disposed alongchannel 322″. In the example depicted in FIG. 12, components representedby double primed reference numbers are configured to perform functionssimilar to the objects represented by corresponding unprimed referencenumbers in FIG. 10. Like air trap 330′, air trap 330″ includes threesections 330 a″, 330 b″, 330 c″. However, rather than including bentangles, the three sections of air trap 330″ are separated from eachother by a pair of narrowed neck regions 332 a″, 332 b″. Like the bentangles of air trap 330′, these neck regions serve to create additionalsites for stopping fluid flow by capillary pinning, to better preventinadvertent flow of sample-containing fluid. Many configurations of airtraps are possible, and the exact configuration may be chosen to resultin a desired amount of resistance to capillary flow. Furthermore,although the term “air trap” has been used, this does not imply that airmust actually be trapped under all circumstances. In some cases, theshape of the air trap may serve to prevent undesirable fluid flow, evenif there is no air trapped in the “air trap.”

FIG. 13 is a semi-transparent top view of another exemplary dropletgenerator, generally indicated at 350, in accordance with aspects of thepresent disclosure. Droplet generator 350 includes a substantiallyplanar substrate 352 having a top surface 354 and a bottom surface 356.A sample well 358 and a background fluid well 360 are integrally formedwith substrate 352. A droplet outlet region, which in this example takesthe form of a pipette tip 362, may be integrally formed with substrate352, or in some cases may be formed separately and integrated with thesubstrate, as described in more detail below. A network of channels,generally indicated at 364, is formed in the bottom surface 356 ofsubstrate 352 and fluidically interconnects the sample well, thebackground fluid well, and the pipette tip.

As used herein, the terms “pipette” and “pipette tip” are not intendedto be limited to the structure shown in FIG. 13 or any of the otherdrawings. More generally, these terms are intended to mean a dropletoutlet region that is capable of conveying droplets from a dropletgenerator to an accumulation vessel, or in some cases a sample inletregion that is capable of conveying sample-containing fluid to a samplewell or a sample inlet channel. A pipette tip can be in the form of achannel. It can be formed separately from a droplet generator housingsuch as a substrate, in which case the pipette tip can mate with thesubstrate to convey droplets from the droplet generator to anothervessel, or to convey sample fluid to the droplet generator. It can alsobe integrally formed with the droplet generator, as in FIG. 13. Anaccumulation vessel suitable for use in conjunction with a pipette canbe any container suitable for accumulating droplets. In particular, theaccumulation vessel can be the well of a microtiter plate or a PCRplate. Pipette tip, as used in this disclosure, can also be a tip thatis used with, e.g., a handheld or automated pipettor.

As in the case of droplet generator 300 described previously, asubstantially planar sealing member (not shown) may be configured to bedisposed adjacent to the bottom surface of substrate 352, to form asubstantially fluid tight seal with the bottom surface of the substrateand thus with channel network 364. The sealing member may be afeatureless planar member, or the network of channels may be partiallyor entirely formed in the sealing member rather than exclusively insubstrate 352. In any case, a fluid tight network of channels will beformed when the substrate and the sealing member are brought together.Furthermore, in some cases, channel network 354 may be integrally formedand/or sealed within substrate 352 in a fluid tight manner, in whichcase there may be no sealing member provided.

Wells 358 and 360 configured to withstand the forces expected whenpressure is applied, when the droplet generator integrated with apumping unit, and when the droplet generator is handled and shipped to acustomer or other destination. Accordingly, wells 358 and 360 may besimilar in their characteristics to previously described wells 308, 310,and 312, i.e., wells 358 and 360 may have thicknesses in the approximaterange from 0.04 to 0.40 inches thick, depending on the expected forcesand the material from which droplet generator 350 is constructed.Similarly, pipette tips 362 will generally be constructed to withstandthese same forces. As mentioned previously, in some cases, pipette tips362 may be integrally formed with substrate 352, for example in aninjection molding process. In other cases, the substrate may be formedwith suitable apertures or other connection structures (not shown inFIG. 13) configured to receive suitably modified, standard pipette tipsin a fluid tight manner.

Channel network 364 defines a droplet generation region indicated at370, which is configured to generate sample-containing dropletssuspended in the background fluid. As in the case of previouslydescribed droplet generation regions 320, each droplet generation region370 is defined by the intersection of a first channel 372 configured totransport sample-containing fluid from sample well 358 to dropletgeneration region 370, a second channel 374 configured to transportbackground fluid from background fluid well 360 to droplet generationregion 370, and a third channel 376 configured to transportsample-containing droplets from droplet generation region 370 to pipettetip 362. Droplets are formed in region 370 according to principles thathave been described in detail above.

Also as described previously, second channel 374 includes two backgroundfluid sub-channels 374 a, 374 b, which intersect first channel 372 fromtwo different directions to form a cross-shaped droplet generationregion. Sub-channels 374 a, 374 b have approximately equal lengths, sothat they have substantially equal hydraulic resistances and the rate ofbackground fluid flow through each sub-channel is substantially thesame. In addition, an air trap 380 is disposed along first channel 372,between sample well 358 and droplet generation region 370, and isconfigured to prevent sample-containing fluid from being inadvertentlydrawn through first channel 372 by capillary action or other forces.Accordingly, droplets will be formed only when suitable pressures areapplied to the sample wells, the background fluid wells, and/or thepipette tips, in which case the formed droplets will be transportedthrough channels 376 to pipette tips 362, and emitted from apertures 382formed in the pipette tips. The emitted sample-containing droplets thenmay be collected and/or further transported for additional processingsteps such as thermocycling.

FIG. 14 is a semi-transparent top view of yet another exemplary dropletgenerator, generally indicated at 350′, in accordance with aspects ofthe present disclosure. Droplet generator 350′ is substantially similarto droplet generator 350 in most respects. Accordingly, primed referencenumbers in FIG. 14 represent substantially the same components as theirunprimed counterparts in FIG. 13, and those components will not bedescribed again here. However, droplet generator 350′ of FIG. 14 differsfrom droplet generator 350 of FIG. 13 in the following respect.

Rather than pipette tips 362 and corresponding apertures 382, thirdchannels 376′ of droplet generator 350′ transport sample-containingdroplets to droplet wells 390′, which collect the droplets in a mannersimilar to droplet wells 312 of droplet generator 300. Thus, dropletgenerators 350 and 350′ may be viewed as slight variations of eachother, with each best suited for a particular application or class ofapplications. Furthermore, these examples show that the droplet wells ofany of the other planar mode droplet generator embodiments describedherein may be replaced with pipette tips under appropriatecircumstances.

FIG. 15 is a top view of still another exemplary planar mode dropletgenerator, generally indicated at 400, in accordance with aspects of thepresent disclosure, and FIG. 16 is a bottom view of droplet generator400. FIG. 17 is a sectional view of droplet generator 400 taken alongthe line 17-17 in FIG. 15. Droplet generator 400 includes the samegeneral components as droplet generator 300, and reference numbersstarting with 400 in FIGS. 15-16 represent substantially the samecomponents as their counterparts starting with 300 in FIGS. 8-10. Asidefrom the features of droplet generator 400 either not included indroplet generator 300 or not discussed in the description of dropletgenerator 300, those components will not be described again here.Several features of droplet generator 400 exemplify features that may beadopted in any of the planar mode droplet generators described herein.Specifically, FIG. 15 depicts background fluid wells 410 and dropletwells 412 as having an oval cross section near the top of each well,whereas sample wells 408 have a circular cross section. An oval shape,as opposed to a circular upper cross section, may facilitate fluid tightconnections between the wells and other components of an overall assaysystem, such as pump interfaces. The use of an oval shape is merelyexemplary. In general, the upper portion of each well may be given anydesired shape in a particular droplet generator, to best facilitate theintegration of the droplet generator with the other portions of theassay system.

In addition, as can be best seen in FIG. 17, one or more of the wells ofa droplet generator according to the present teachings, in this casedroplet wells 412, may have a stepped vertical cross section in whichthe well becomes narrower toward the bottom in a stepped fashion. On theother hand, also as depicted in FIG. 17, other wells, such as samplewells 408 and background fluid wells 410, may have smoothly taperedvertical cross sections, which also become narrow toward the bottom ofthe wells. The use of stepped and/or smoothly tapered vertical wellcross sections may facilitate the manufacture of injection molded planarmode droplet generators.

The depicted cross sections also may have other advantages, such as thefollowing. The steps or other features in the well bottoms can guide afluid dispenser, such as a pipette tip, to a position in the well thatis optimal for liquid transfer in and out of the device. Without suchfeatures, a pipette tip could, for instance, be inserted such that,during liquid addition, the liquid is injected directly into thechannels. Likewise, features in a droplet well can allow a pipette tipto be conveniently positioned a fixed distance from the well bottom,allowing droplet to be aspirated without being damaged while flowingthrough a “pinch” between the pipette tip and well bottom.

Smoothly tapered well walls may help to facilitate drainage of thesample toward the well bottoms, which leaves less residual sample in thewell, and increases the efficiency of sample conversion to droplets.Samples are often precious, and high sample conversion efficiency is avaluable feature. The use of smoothly tapered walls may result in asample loss of less than 0.5 uL, or even less than 0.3 nL. For a 20 uLsample, the conversion efficiency is then over 95%.

FIG. 16 illustrates a network of fluid channels 414 that differsslightly from its counterpart network 314 of droplet generator 300.Specifically, first channel 422, which is configured to transportsample-containing fluid from sample well 408 to droplet generationregion 420, does not include an air trap. As described previously, anair trap may be used in some cases to help prevent unwanted fluidtransport from the sample well to the droplet generation region.However, as depicted in FIG. 16, an air trap may not always benecessary. This may be the case, for example, if the channel between thesample well and the droplet generation region is given a hydraulicresistance sufficient to prevent unwanted fluid flow. As depicted forchannel 422 of droplet generator 400, this may be accomplished if thechannel has sufficient length, a great enough number of bends, a smallenough diameter, and/or is given other characteristics (such as anappropriately coated inner surface) to raise its hydraulic resistance toa desired level.

Aside from giving channel 422 hydraulic resistance sufficient to avoidunwanted transport of sample-containing fluid, FIG. 16 also shows howthe various channels of network 414 may be given hydraulic resistancesresulting in a desired rate of droplet production. More specifically,each of channels 422, 424 (including sub-channels 424 a, 424 b), and 426may be configured to have any desired hydraulic resistance, by givingthose channels desired lengths and/or other suitably chosencharacteristics. In this manner, the overall hydraulic resistance ofchannel network 414 may be tuned to any desired level, to result in apredetermined flow rate of sample-containing droplets into droplet wells412 for a given set of applied pressures. These same principles may beapplied to any droplet generation system according to the presentteachings.

FIGS. 18, 19, and 24 depict a first example of a planar mode dropletgenerator, generally indicated at 500, in which a planar substrate and awell vessel are formed as separate components. A benefit of this designis that a variety of well sizes can be used with a single dropletgenerator base. It also permits replacement of, e.g., the sample and oilwells with syringe pumps containing those liquids. This enables, forinstance, “bulk” droplet generation on the mL scale versus the typicaluL scale.

FIG. 18 is a top view of droplet generator 500, and FIG. 19 is amagnified perspective view of a portion of the droplet generator.Droplet generator 500 includes a substantially planar substrate 502having a top surface 504 and a bottom surface 506, and a separate wellvessel 508 configured to be connected to substrate 502 to form afunctional droplet generator. Although in the depicted examples, all ofthe fluid wells associated with the droplet generator are formed in awell vessel, in alternative configurations contemplated by the presentteachings, one or more of the sample well, background fluid well(s), anddroplet well may be integrally formed with the substrate (as inpreviously described embodiments), while the remainder of the wells areincluded in the well vessel.

To accommodate a connection with well vessel 508, top surface 504 ofsubstrate 502 includes various ports configured to receive complementaryportions of the well vessel. Specifically, a sample port 510, a pair ofbackground fluid ports 512 a, 512 b, and a droplet outlet port 514 areall formed in top surface 504 of substrate 502. In droplet generator500, each of these ports takes the form of a substantially cylindricalaperture, but any desired shape may be used for the ports. Well vessel508 includes a sample well 516 configured to make a substantially fluidtight connection with sample port 510, a pair of background fluid wells518 a, 518 b configured to make a substantially fluid tight connectionwith background fluid ports 512 a, 512 b, and a droplet outlet well 520configured to make a substantially fluid tight connection with dropletoutlet port 514.

To accomplish a fluid tight connection between each well and itsassociated port, each well includes a cylindrical attachment protrusion522 configured to fit securely and in a fluid tight manner within thecorresponding port. When the ports are given shapes other thancylindrical, the attachment protrusions of the well vessel will be givenappropriate complementary shapes. Furthermore, according to the presentteachings, the attachment between the ports and the wells may be made inmany different ways. For example, in contrast to the depiction of FIGS.18-19, the “male” portions of the attachment mechanisms (i.e., theprotrusions) may be associated with the ports, and the “female” portionsof the attachment mechanisms (i.e., the complementary apertures) may beassociated with the wells, for some or all of the wells. In any case,the ports and/or the wells may be provided with various elements such aso-rings, compression plates, or elastic apertures, to facilitate a fluidtight connection between the substrate and the well vessel. Forinstance, a short length of Tygon elastic tubing, sold by theSaint-Gobain Corporation of France, may fit snugly on the outsides ofthe “male” portions of any attachment mechanisms.

FIG. 24 depicts the bottom surface of the droplet generator, with apossible sealing member (described below) removed for clarity. Asdepicted in the central portion of FIG. 24, a network of channels,generally indicated at 530, is formed in the bottom surface 506 ofsubstrate 502. Network 530 is configured to fluidically interconnectsample port 510, background fluid ports 512 a, 512 b, and droplet outletport 514. A droplet generation region 532 is defined by network ofchannels 530 and configured to generate sample-containing dropletssuspended in the background fluid. More specifically, droplet generationregion 532 is defined by the intersection of a sample channel 534, apair of background fluid channels 536 a, 536 b, and droplet channel 538.Sample channel 534 is configured to transport sample-containing fluidfrom sample port 510 to droplet generation region 532, background fluidchannels 536 a, 536 b are respectively configured to transportbackground fluid from background fluid ports 512 a, 512 b to dropletgeneration region 532, and droplet channel 538 is configured totransport sample-containing droplets from droplet generation region 532to droplet outlet port 514.

As depicted in FIG. 19, a substantially planar sealing member 540 may beprovided with droplet generator 500. As in the case of the other planarmode embodiments, sealing member 540 may be attached to substrate 502 bycompression, adhesion, heat sealing, or any other suitable attachmentmechanism, to make channel network 530 fluid tight. Also as describedpreviously, in some cases the channel network may be formed partially orentirely in the sealing member rather than in the bottom surface of thesubstrate. Furthermore, any other desired features may be introducedinto channel network 530, such as increased channel lengths, changes inchannel diameter and/or cross section, or an air trap disposed betweenthe sample port and the droplet generation region, to control the timingand rate of droplet generation as described above.

FIGS. 20-21 depict another example of a planar mode droplet generator,generally indicated at 550, in which a planar substrate and a wellvessel are formed as separate components. FIG. 20 is a top view ofdroplet generator 550, and FIG. 21 is a magnified perspective view of aportion of the droplet generator. Droplet generator 550 is similar inmany respects to droplet generator 500 depicted in FIGS. 18-19, andaccordingly some of the common features of droplet generator 550 anddroplet generator 500 will not be described again in detail.

Droplet generator 550 includes a substantially planar substrate 552having a top surface 554 and a bottom surface 556, and a separate wellvessel 558 configured to be connected to substrate 552 to form afunctional droplet generator. In this example, however, well vessel 558has only three wells, rather than four as in the case of dropletgenerator 500. To accommodate a connection with well vessel 558, topsurface 554 of substrate 552 includes ports configured to receivecomplementary portions of the well vessel. Specifically, a sample port560, a background fluid port 562, and a droplet outlet port 564 are allformed in top surface 554 of substrate 552. Each of these ports takesthe form of a substantially cylindrical aperture, but any desired shapemay be used.

Well vessel 558 includes a sample well 566 configured to make asubstantially fluid tight connection with sample port 560, a backgroundfluid well 568 configured to make a substantially fluid tight connectionwith background fluid port 562, and a droplet outlet well 570 configuredto make a substantially fluid tight connection with droplet outlet port564. As in the case of droplet generator 500, regardless of the shapesof the ports, each well includes a complementary protrusion 572configured to fit securely and in a fluid tight manner within thecorresponding port. Furthermore, the connection between the ports andthe wells may be made in many different ways, and may include variouscomponents configured to facilitate a fluid tight connection, asdescribed previously with respect to droplet generator 500.

As depicted in FIG. 21, a substantially planar sealing member 574 may beprovided with droplet generator 550. As in the case of the other planarmode embodiments, sealing member 574 may be attached to substrate 552 byany suitable attachment mechanism. Also as described previously, anetwork of channels (not shown) may be formed in the substrate, or maybe formed partially or entirely in the sealing member, to fluidicallyinterconnect the sample port, the background fluid port, and the dropletoutlet port. This network of channels will generally provide a dropletgeneration region configured to generate sample-containing droplets inthe background fluid, and may include any suitable characteristics ofthe other channel networks described herein.

FIGS. 22-24 depict yet another example of a planar mode dropletgenerator, generally indicated at 600, in which a planar substrate and awell vessel are formed as separate components. FIG. 22 is a top view ofdroplet generator 600, and FIG. 23 is a magnified perspective view of aportion of the droplet generator. FIG. 24 depicts the bottom surface ofthe droplet generator, with a planar sealing member removed for clarity.Droplet generator 600 is substantially similar to droplet generator 550depicted in FIGS. 20-21 in some respects, and accordingly some of thesimilarities between droplet generator 600 and droplet generator 550will not be described in detail below.

Droplet generator 600 includes a substantially planar substrate 602having a top surface 604 and a bottom surface 606, and a separate wellvessel 608 configured to connect to substrate 602. As in the case ofdroplet generator 550, well vessel 608 has only three wells, rather thanfour as in the case of droplet generator 500. Top surface 604 ofsubstrate 602 includes a sample port 610, a background fluid port 612,and a droplet outlet port 614, all formed in top surface 604 ofsubstrate 602. Each of these ports takes the form of a substantiallycylindrical aperture, but as described previously, any desired shape maybe used.

Well vessel 608 includes a sample well 616, a background fluid well 618,and a droplet outlet well 620, each configured to make a substantiallyfluid tight connection with the associated ports of the substrate. As inthe case of droplet generators 500 and 550, regardless of the shapes ofthe ports, each well includes a complementary protrusion 622 configuredto fit securely and in a fluid tight manner within the correspondingport. In addition, the connection between the ports and the wells may bemade in many different ways, and may include various componentsconfigured to facilitate a fluid tight connection, as describedpreviously with respect to droplet generators 500 and 550.

The primary difference between droplet generator 600 and dropletgenerator 550 is the spacing between sample well 616 and backgroundfluid well 618. As depicted in FIGS. 22-23, in the case of dropletgenerator 600, these wells are not configured to fit into adjacent portsof the substrate as in the case of droplet generator 550, but rather areconfigured to fit into ports that are separated from each other byanother, unused port. This spacing provides certain possible advantagesto the droplet generator, as described below.

As depicted in the non-central portions of FIG. 24, a network ofchannels, generally indicated at 630, is formed in the bottom surface606 of substrate 602. Network 630 is configured to fluidicallyinterconnect sample port 610, background fluid port 612, and dropletoutlet port 614. A droplet generation region 632 is defined by networkof channels 630 and configured to generate sample-containing dropletssuspended in the background fluid. More specifically, droplet generationregion 632 is defined by the intersection of a sample channel 634, apair of background fluid sub-channels 636 a, 636 b, and droplet channel638. Sample channel 634 is configured to transport sample-containingfluid from sample port 610 to droplet generation region 632, backgroundfluid sub-channels 636 a, 636 b are configured to transport backgroundfluid from background fluid port 612 to droplet generation region 632,and droplet channel 638 is configured to transport sample-containingdroplets from droplet generation region 632 to droplet outlet port 614.

As FIG. 24 indicates, background fluid sub-channels 636 a, 636 b areeach fed by a background fluid channel 636 that transports backgroundfluid from background fluid well 618. Sub-channels 636 a, 636 b thentransport background fluid to droplet generation region 632 so that thebackground fluid arrives at the droplet generation region from twodifferent directions, forming a cross-shaped (or topologicallyequivalent) droplet generation region. As has been described withrespect to previous planar droplet generator embodiments, sub-channels636 a, 636 b may be configured to have substantially the same hydraulicresistance, for example by providing the sub-channels with substantiallythe same lengths, so that background fluid reaches droplet generationregion 632 with substantially the same flow rate in each sub-channel.

In addition, other features may be provided to channel network 630,including an air trap 640 disposed along the path of thesample-containing fluid, and regions of varying channel diameter, asindicated by the diameter of sub-channels 636 a, 636 b in the vicinityof droplet generation region 632. Furthermore, a planar sealing member642 may be provided, which in some cases may include all or a portion ofthe channel network. These features serve purposes that have beendescribed previously.

IV. Continuous Mode Examples

This section describes examples of “continuous mode” droplet generators,in which the droplet generator is manufactured from a single piece ofmaterial; see FIGS. 7 and 25-33. As described in more detail below withrespect to several specific examples, an advantage of this single piecedesign is that there is no need to precisely align multiple parts toform the droplet generator geometry.

One type of continuous mode droplet generator is based on a hollow tubewith one or more holes drilled through the tube walls to intersect thehollow channel. For example, FIG. 7, which has been describedpreviously, depicts several different droplet generator geometries ofthis type. Specifically, droplet generators 200, 220, and 240 all may becharacterized as continuous mode droplet generator tubes, because theyare formed from a single piece of material. In contrast, dropletgenerator 260 of FIG. 7 is formed of two separate butted tubes requiringcareful alignment, and thus would not be characterized as a continuousmode droplet generator.

FIG. 25 depicts yet another type of continuous mode droplet generator,generally indicated at 700, which is similar in some respects to dropletgenerators 200, 220, and 240 of FIG. 7. Droplet generator 700 includes ahollow droplet generator tube 702 having a channel 704 running along itslength. A slit 706 is partially cut into the side of the tube, tointersect channel 704 and divide the channel into two aligned sections704 a and 704 b. Slit 706 also forms a radial flow channel for the flowof background fluid. Accordingly, if sample-containing fluid istransported through channel section 704 a and background fluid istransported into slit 706, sample-containing droplets may be created andsuspended in the background fluid, and the resulting emulsion may betransported through channel section 704 b.

FIGS. 26-27 depict an example of a more complete continuous mode dropletgenerator system, generally indicated at 750. FIG. 26 is an explodedelevational view of droplet generator system 750, and FIG. 27 is anassembled elevational view of the droplet generator system. Dropletgenerator system 750 includes a sample well 752, a tubular dropletgenerator 754, an oil feed connector 756, and a housing base 758, allconfigured to fit securely into a droplet generation housing 760. Acompression plate 762 is used to compress housing base 758 againsthousing 760, to form a fluid tight surface at the bottom of the housing.Compression is caused by a pair of compression screws 764 a, 764 b,which fit into corresponding apertures 766 a, 766 b in the compressionplate. Aligned apertures (not shown) in the housing base and the housingreceive the compression screws and allow them to compress the housingbase into the housing.

A segment of hollow, stainless steel tubing 768 fits within alignedcentral apertures 770, 772 of the housing base and compression plate,respectively, and extends into housing 760. Droplet generator 754 may beinserted through tubing 768, into the interior of housing 760, and intoa distal aperture 773 of sample well 752. More specifically, the dropletgenerator is inserted into the distal aperture of sample well 752 toform a (disposable) sample handling assembly. The sample handlingassembly is inserted into the (non-disposable) housing assembly. Afteruse, the sample handling assembly may be contaminated with sample andcan be discarded. A sample handling assembly may be used with eachsample to reduce cross-contamination.

In this example, sample well 752 includes a Luer taper 774 configured tofit into a corresponding “female” Luer mating portion of housing 760, toform a substantially leak-free connection between the sample well andthe housing. Aperture 773 is disposed at the distal end of taper 774,and is configured to securely receive droplet generator 754. Similarly,oil feed connector 756 also may include a Luer taper (not shown),configured to fit into a corresponding aperture of housing 760, whichthus provides a background fluid input channel for oil or some otherbackground fluid to enter the housing.

Sample well 752 also includes a reservoir portion 776 configured toreceive sample-containing fluid to be used in forming sample-containingdroplets. A proximal aperture 778 of the reservoir portion may beconfigured to receive standardized or proprietary fluid fittings and/orpressure fittings. This may facilitate the transfer of sample-containingfluid to the sample well, and/or the application of pressure to thesample-containing fluid to cause the formation of sample-containingdroplets. Similarly, a proximal aperture 779 of the oil feed connectormay be configured to accept standard or proprietary fluid fittingsand/or pressure fittings, to facilitate the transfer of pressurized oilor some other background fluid into housing 760.

Droplet generator 754 may be similar to any of the previously describeddroplet generator tubes, such as tubes 200, 220, 240, or 700. Morespecifically, in this example droplet generator 754 is a continuoushollow tube having a slit 780 formed at an intermediate location alongthe length of the tube. Slit 780 is oriented substantially normallyrelative to the length of the droplet generator tube, and extends farenough into the droplet generator tube to intersect the central channelof the tube. More generally, slits and/or channels that penetrate fromthe outer periphery of the droplet generator to intersect its centralchannel may be oriented at any desired angle(s). Furthermore, theseslits and/or channels need not pass linearly from the periphery of thedroplet generator tube toward its central channel, but may be configuredto have any desired trajectories. This may, for example, allow thehydraulic resistance of the background fluid channel to be tuned to adesired value, as a manner of controlling the rate of production ofsample-containing droplets.

When droplet generator 754 is inserted into tubing 768 and passesthrough the interior of housing 760 and into distal aperture 773 ofsample well 752, slit 780 will be exposed to any fluid present in theinterior portion of housing 760. When sample-containing fluid istransported from sample well 752 into droplet generator tube 754, thesample-containing fluid eventually reaches slit 780, where it encounterspressurized background fluid that has been transported into housing 760via the background fluid input channel of the housing. Sample-containingdroplets suspended in the background fluid are created in the vicinityof the slit, and transported further down the droplet generator, wherethey eventually reach a droplet outlet region 782 defined by the distalend of the droplet generator tube.

FIGS. 28-29 depict another example of a continuous mode dropletgeneration system, generally indicated at 800. Droplet generation system800 includes a sample well 802, a droplet generator 804 configured toreceive sample-containing fluid from the sample well, and an outerhousing 806 configured to selectively receive the droplet generator. Insome cases, the sample well and the droplet generator, which come intodirect contact with sample-containing material, may be configured as adisposable component, whereas the housing, which does not come intodirect contact with sample-containing material, may be configured as areusable component. The droplet generator may be integrally formed withthe sample well, or it may be formed separately and selectivelyintegrated with the sample well.

In system 800, droplet generator 804 takes the form of a dropletgeneration tube, of a type described previously. More specifically,droplet generator 804 is a continuous hollow tube 808 having a slit 810formed at an intermediate location along the length of the tube. One endof the droplet generator is in fluidic communication with sample well802. This may occur during formation of the sample well and dropletgenerator, if they are integrally formed, or if the droplet generatorand sample well are formed separately, the droplet generator may beselectively placed in fluid communication with the sample well, bypositioning it securely against a lower outlet aperture of the well. Theother end of the droplet generator defines a droplet outlet region 812configured to receive sample-containing droplets generated within adroplet generation region of the droplet generator. The dropletgenerator and the sample well are disposed within a substantiallyfrustoconical inner housing 814, which is configured to fit securelywithin a corresponding aperture 816 in outer housing 806.

Frustoconical inner housing 814 is sized so that sample well 802 will bedisposed at or above the upper surface 818 of housing 806, while dropletoutlet region 812 will be disposed at or below the lower surface 820 ofhousing 806. Inner housing 814 includes a slit 822, which exposes slit810 of droplet generator tube 808 to any fluid that penetrates slit 822.When inner housing 814 is properly disposed within housing 806, slits822 and 810 will be aligned with a pair of background fluid inputchannels 824 a, 824 b which are formed within housing 806 and configuredto provide background fluid to the droplet generator from a backgroundfluid source. A pair of elastic o-rings 826, or other suitablecomponents, may be used to secure inner housing 814 within outer housing806 in a leak-proof manner and at the proper location. Whensample-containing fluid is transported from sample well 802 into dropletgenerator 804, it eventually reaches the region of intersection of slit810 and background fluid input channels 824 a, 824 b, at which pointsample-containing droplets suspended in the background fluid aregenerated and directed toward droplet outlet region 812, where they maybe collected and/or transported for subsequent assay steps.

FIGS. 30-31 depict still another example of a continuous type dropletgeneration system, generally indicated at 850. FIG. 30 is a schematicelevational view of system 850, and FIG. 31 is a magnified sectionalview of a portion of the system. Droplet generation system 850 includesa sample well 852 and an integrated droplet generator 854, whichcollectively take the form of a modified pipette as generally indicatedat 856. Droplet generation system 850 also includes a housing 858configured to selectively receive pipette 856.

Pipette 856 includes a central channel 860, which transportssample-containing fluid from the sample well portion 852 of the pipette,downward in FIGS. 30-31. Droplet generator 854 is provided in pipette856 by forming a pair of horizontal channels 862 a, 862 b in the pipettetip, which intersect central channel 860. Thus, droplet generator 854 issimilar to previously described examples in which a droplet generator isformed from a hollow tube that includes channels extending from theperiphery of the pipette to its central channel. In other cases, thedroplet generator of the current example can be formed using a slit thatextends from the periphery of the pipette to its central channel, as hasbeen described previously.

To receive the pipette, housing 858 includes a cavity, generallyindicated at 860, which consists of a first, tapered bore section 864and a second, cylindrical bore section 866. Pipette 856 fits securelywithin the tapered bore section, leaving a small amount of open spacearound a portion of the pipette disposed within the cylindrical boresection, in the vicinity of channels 862 a, 862 b. Housing 858 alsoincludes a horizontal bore 868, which intersects cylindrical boresection 866 and extends beyond it. Adjacent to horizontal bore 868 (onthe left-hand side of FIG. 31) is an internally threaded aperture 870,which is configured to receive a background fluid input device 872.

The background fluid input device can be used to provide pressurizedbackground fluid to horizontal bore 868. Background fluid provided bythe fluid input device will fill horizontal bore 868 and cylindricalbore section 866, and enter horizontal channels 862 a, 862 b of thedroplet generator, where it will intersect sample-containing fluidpassing downward through channel 860. According to previously describedprinciples, an emulsion of sample-containing droplets suspended in thebackground fluid will therefore be produced, and will travel furtherdown channel 860 until they reach a droplet outlet region 874 defined bya distal aperture of pipette 856.

FIG. 32 depicts an exploded view of yet another example of a continuoustype droplet generation system, generally indicated at 900. Dropletgenerator 900 includes a sample well 902, a droplet generator 904, and ahousing 906. Sample well 902 includes a conical interior portion 908configured to hold sample-containing fluid. An aperture 910 allows fluidto pass from the sample well into droplet generator 904, as described inmore detail below. Droplet generator 904 is a tubular droplet generatorof a type described previously, including a central fluid channel 912and a transverse slit 914 that intersects the central channel. Dropletgenerator 904 is configured to fit securely within a complementaryaperture (not shown) formed in the bottom surface of sample well 902, sothat the droplet generator will be fluidically connected with the samplewell, with a portion of the droplet generator (including slit 914)extending below the sample well.

Housing 906 includes a central aperture 916 configured to receive samplewell 902, and a cylindrical bore 918 extending below the centralaperture and configured to receive a lower portion of droplet generator904. A plurality of alignment features 920 are provided in the interiorof central aperture 916 and configured to align sample well 902 anddroplet generator 904 in desired positions within housing 906. Morespecifically, when sample well 902 and droplet generator 904 are alignedcorrectly within housing 906, slit 914 of the droplet generator will bedisposed below alignment features 920 and above cylindrical bore 918. Atthe same time, the upper portion of sample well 902 will beapproximately aligned with the upper portion of housing 906, although insome cases the upper portions of well 902 and housing 906 may be offsetby a desired predetermined amount.

Housing 906 is configured to receive a background fluid such as oil, andthus to function as a background fluid well. Specifically, backgroundfluid may be disposed at least in the portion of central aperture 916below alignment features 920 and above cylindrical bore 918.Accordingly, when system 900 is assembled so that sample well 902 anddroplet generator 904 are aligned correctly within housing 906, slit 914of droplet generator 904 will be submerged in background fluid. Apressure source (not shown) then may be connected to the upper surfaceof housing 906, and in some cases also to the upper surface of samplewell 904, to apply pressure to both the sample-containing fluid in thesample well and the background fluid in the housing.

Upon application of pressure from a pressure source, thesample-containing fluid and the background fluid will intersect at adroplet generation region defined by the intersection of central channel912 and slit 914, and sample-containing droplets suspended in thebackground fluid will be generated. This emulsion of droplets will thenbe transported through the lower portion of droplet generator 904,toward a droplet outlet region 922 defined by the distal outlet of thedroplet generator. From there, the sample-containing droplets may becollected and/or transported for a subsequent assay step such asthermocycling.

FIGS. 33-34 depict an exemplary multi-sample continuous mode dropletsystem, generally indicated at 950. FIG. 33 shows system 950 in anassembled state, and FIG. 34 is an exploded view of a portion of system950. Droplet generator system 950 includes a droplet generator assembly952, and a droplet well plate 954 configured to receivesample-containing droplets that have been generated by the dropletgenerator assembly.

Droplet generator assembly 952 is similar in some respects to dropletgenerator system 750 depicted in FIGS. 26-27. Specifically, dropletgenerator assembly 952 includes a plurality of sample wells, generallyindicated at 956, each with an associated tubular droplet generator 958,an oil feed connector 960, and a housing base 962, all configured to fitsecurely within a housing 964. A compression plate 966 is used tocompress housing base 962 against housing 964, to form a fluid tightsurface at the bottom of the housing. The compression plate may, forexample, utilize compression screws (not shown) to compress the housingbase against the housing, or it may be preloaded and then adhered to thehousing in any suitable manner. Gasket seals 968 having centralapertures 970 are disposed between housing base 962 and compressionplate 966, to further prevent leakage from the housing.

Segments of hollow, stainless steel tubing 972 fit within alignedcentral apertures 970, 974, 976 of the gasket seals, the housing baseand the compression plate, respectively, and tubing segments 972 extendpartially into housing 964. When system 950 is assembled, dropletgenerators 978 extend through tubing segments 972, into the interior ofhousing 964, and into distal apertures 980 of sample wells 956. Samplewells 956 may include Luer tapers 982 configured to fit into acorresponding “female” Luer mating portion of housing 964, to form asubstantially leak-free connection between the sample well and thehousing. Apertures 980 are disposed at the distal ends of tapers 982,and are configured to securely receive droplet generators 978.Similarly, oil feed connector 960 also may include a Luer taper 984,configured to fit into a corresponding aperture of housing 964, andthereby to provide a background fluid input channel for oil or someother background fluid to enter the housing.

Sample wells 956 also include reservoir portions, generally indicated at986, configured to receive sample-containing fluid to be used in formingsample-containing droplets. A proximal aperture 988 of each reservoirportion may be configured to receive standardized or proprietary fluidfittings and/or pressure fittings. This may facilitate the transfer ofsample-containing fluid to the sample well, and/or the application ofpressure to the sample-containing fluid to cause the formation ofsample-containing droplets. Similarly, a proximal aperture 990 of theoil feed connector may be configured to accept standard or proprietaryfluid fittings and/or pressure fittings, to facilitate the transfer ofpressurized oil or some other background fluid into housing 964.

Droplet generators 978 may be similar to any of the previously describeddroplet generator tubes, such as tubes 200, 220, 240, or 700. Morespecifically, in this example, droplet generators 978 each take the formof a continuous hollow tube having a slit 992 formed at an intermediatelocation along the length of the tube. Slits 992 extend far enough intothe associated droplet generator tube to intersect the central channelof the tube. When droplet generators 978 are inserted into tubingsegments 972 and pass through the interior of housing 964 and intodistal apertures 980 of sample wells 956, each slit 992 will be exposedto background fluid present in the interior portion of housing 964.

Thus, when sample-containing fluid is transported from each sample well956 into droplet generator tubes 978, the sample-containing fluideventually reaches a slit 992, where it encounters pressurizedbackground fluid that has been transported into housing 964 via thebackground fluid input channel of the housing. Sample-containingdroplets suspended in the background fluid are created in the vicinityof the slit, and transported further down the droplet generator, wherethey eventually reach one of droplet outlet regions defined by thedistal end of the associated droplet generator tube.

V. Two-Part Mode Examples

This section provides examples of two-part mode droplet generationsystems, in which a first portion of the system contains a samplechannel for transporting sample-containing fluid to a droplet generationregion, and a second portion of the system contains a droplet channelfor transporting sample-containing droplets away from the dropletgeneration region; see FIGS. 35-42. A background fluid channel fortransporting background fluid to the droplet generation region may beincluded with either the first or second portions of the system, or maybe included in a separate portion. In some cases, the first portion ofthe system, which comes into direct contact with the sample-containingfluid, may be configured as a disposable component, and the secondportion of the system, which does not come into direct contact with thesample-containing fluid, may be configured as a reusable component.Furthermore, the term “two-part” is not meant to be limiting; in somecases, systems according to this mode may use three or more separatecomponents.

FIGS. 35-38 depict a first example of a two-part droplet generationsystem, generally indicated at 1000, in accordance with aspects of thepresent teachings. As depicted in FIG. 35, system 1000 includes asubstantially planar droplet generator substrate 1002, and a samplecontainer, which in this example takes the form of a pipette tip 1004.Substrate 1002 includes a droplet generation well 1006, and an emulsionwell 1008. A droplet channel 1010 formed in the substrate fluidicallyinterconnects the droplet generation well and the emulsion well. Furtherdetails of substrate 1002 and pipette tip 1004 are shown in FIGS. 36-38.

FIG. 36 is a magnified sectional view of an end portion of pipette tip1004. As FIG. 36 depicts, pipette tip 1004 includes a sample wellportion 1012, and a sample channel 1014 through which sample-containingfluid may be transported from sample well portion 1012 to dropletgeneration well 1006 of substrate 1002.

FIG. 37 is a magnified sectional view of droplet generation well 1006,and FIG. 38 is a top view of droplet generation well 1006. As shown inFIG. 38, the droplet generation well includes an upper well portion1016, and a plus-shaped lower well portion 1018. An emulsion outletchannel 1020, formed in substrate 1002, fluidically interconnects lowerwell portion 1018 with droplet channel 1010. To seal channels 1010 and1020, and thus to provide a leak-free fluid channel between dropletgeneration well 1006 and emulsion well 1008, a sealing member 1026 maybe disposed along the bottom surface of substrate 1002. Sealing member1026 may, for example, take the form of a flexible film that may beadhered to the bottom of the substrate, or it may be a relativelyinflexible member that is constructed from a material similar to thematerial of the substrate itself, such as a thermoplastic material. Inthe latter case, all or a portion of channel 1010 may be formed insealing member 1026 rather than in substrate 1002.

To generate an emulsion of sample-containing droplets suspended inbackground fluid such as oil, sample-containing fluid is loaded intosample well portion 1012 of pipette tip 1004, and background fluid isloaded into droplet generation well 1006. A distal end portion 1022 ofpipette tip 1004 is then placed into the droplet generation well,partially within plus-shaped lower well portion 1018. The outer diameterof distal end portion 1022 is small enough to fit within the upperopening of plus-shaped lower well portion 1018, but too large to fitwithin the lower outlet of the plus-shaped well portion, due to thepresence of step-like features 1019. Thus, when inserted fully intodroplet generation well 1006, the pipette tip rests on top of step-likefeatures 1019, but oil can pass around the outer periphery of endportion 1022 to reach outlet 1024 of sample channel 1014 of the pipettetip. Furthermore, step-like features 1019 can be given any desiredthickness, to space distal end portion 1022 of the pipette tip anydesired distance from emulsion outlet channel 1020.

Accordingly, pipette tip 1004 and emulsion outlet channel 1020 form abutted tube type droplet generator, with the gap between pipette tip1004 and channel 1020 set by the depth of plus-shaped lower well portion1018. See, e.g., droplet generator 280 of FIG. 7 and the accompanyingdiscussion above. To form an emulsion, negative pressure is applied toemulsion well 1008, drawing sample-containing fluid out of pipette tip1004 and also drawing background fluid out of droplet generation well1006. Sample-containing droplets are formed as the sample-containingfluid and the background fluid each pass through plus-shaped lower wellportion 1018. The resulting emulsion then passes through emulsion outletchannel 1020 and droplet channel 1010, to reach emulsion well 1008.

FIGS. 39-40 depict another example of a two-part droplet generationsystem, generally indicated at 1050, in accordance with aspects of thepresent teachings. FIG. 39 is an isometric view of system 1050, and FIG.40 is a sectional view taken along the line 40-40 in FIG. 39. System1050 includes an input housing 1052, a droplet generator housing 1054,which also may be referred to as a sample cartridge, and an outputhousing 1056. Input housing 1052 includes structures such as threadedapertures for receiving a sample pressure source 1058 and a backgroundfluid source 1060; a threaded aperture 1062 for receiving pressuresource 1058 can be seen in FIG. 40. A lower aperture 1064 is formed atthe bottom surface of input housing 1052, and configured to receive anupper portion of droplet generator housing 1054. Sample reservoir 1066is configured to be in fluid communication with the distal end portionof sample pressure source 1058.

Input housing 1052 also includes a background fluid channel, generallyindicated at 1068, which is configured to transport background fluidfrom background fluid source 1060, through the input housing, and intodroplet generator housing 1054. Specifically, background fluid channel1068 includes a first sub-channel 1068 a configured to transportbackground fluid from background fluid source 1060 within input housing1052, to a second sub-channel 1068 b configured to transport backgroundfluid from input housing 1052 into sub-channel 1068 c of dropletgenerator housing 1054.

Droplet generator housing 1054 includes a background fluid channel 1068c that serves as a continuation of channel 1068 b when input housing1052 is aligned correctly with droplet generator housing 1054. Dropletgenerator housing 1054 also includes a hollow cylinder 1070, and a loweraperture 1072. In some cases, cylinder 1070 may be integrally formedwith droplet generator housing 1054. In other cases, as depicted in FIG.40, the droplet generator may include a cylindrical bore 1074 configuredto receive cylinder 1070. In either case, an axial sample-containingfluid channel 1076 of cylinder 1070 will be placed into contact with thedistal end portion of sample reservoir 1066 when system 1050 isassembled.

Output housing 1056 includes a stepped cylindrical aperture 1078.Aperture 1078 is configured to receive a lower, outer portion of dropletgenerator housing 1054, in such a manner that a fluid tight seal isformed between the droplet generator and the output housing.Furthermore, when system 1050 is assembled, sample-containing fluidchannel 1076 of cylinder 1070 will be in substantial alignment with adroplet outlet channel 1080 formed in output housing 1056. Accordingly,cylinder 1070 and output housing 1056 form a butted tube style dropletgenerator, as has been described previously.

An emulsion of sample-containing droplets suspended in a backgroundfluid such as oil is generated with system 1050 as follows. Sample isplaced in sample reservoir 1066. The system 1050 is assembled. Oil orsome other background fluid is supplied via background fluid source1060, which partially fills a lumen space between lower aperture 1072and cylinder 1070. A pressure is supplied via pressure source 1058,causing sample to flow to droplet generation region 1082. Droplets arecollected via droplet outlet channel 1080. The sample contactingportions of system 1050, including hollow cylinder 1070 and housing1054, are configured to be disposable after creating an emulsion.

FIGS. 41-42 depict still another example of a two-part mode dropletgeneration system, generally indicated at 1100. Droplet generationsystem 1100 includes a droplet generator plate, generally indicated at1102, and a removable sample module 1104. In some cases, the dropletgenerator plate will be configured as a reusable component, whereas thesample module will be configured as a disposable component.

Droplet generator plate 1102 includes a substantially planar substrate1106 having a pair of background fluid channels 1108, 1110 extendingfrom opposite sides of the substrate toward the center of the substrate.More specifically, each background fluid channel includes a respectivepair of sub-channels 1108 a, 1108 b and 1110 a, 1110 b, where one of thesub-channels on each side is parallel to the planar top and bottomsurfaces of substrate 1106, and the other sub-channel on each side isnormal to the planar surfaces of the substrate. Each of the verticalsub-channels 1108 b, 1110 b is in fluid communication with a horizontalbackground fluid channel 1112 formed in substrate 1106, which spans thegeometric center of the substrate and intersects a vertical dropletoutlet channel 1114 that is also formed in the substrate. Dropletgenerator plate 1102 further defines a central cylindrical bore 1115,configured to receive a cylindrical sample tube as described below.

Sample module 1104 includes a sample well portion 1116 formed in anupper portion of the sample module, which provides sample-containingfluid to a vertical sample fluid channel 1118. Sample fluid channel 1118may be formed in a cylindrical tube 1120 inserted into or integrallyformed with the sample module, and which extends a predetermineddistance below a bottom surface 1122 of sample module 1104. Thisdistance is determined by the thickness of a plus-shaped spacing feature1123 of the sample module. Tube 1120 is unable to fit through theaperture defined by plus-shaped feature 1123, and thus stops when itcontacts the plus-shaped feature. As a result, sample tube 1120 extendsinto central cylindrical bore 1115 so that a slight gap is left betweenthe bottom of tube 1120 and the top of droplet outlet channel 1114, toform a butted tube type droplet generator defined by a dropletgeneration region, generally indicated at 1124, where background fluidtransported by channel 1112 intersects with sample fluid transported bychannel 1118.

To form an emulsion of sample-containing droplets with system 1100,sample-containing fluid is placed in sample well 1116, and sample module1104 is assembled with droplet generator plate 1102. Background fluid istransported into droplet generator plate 1102 from each side, andpressure is applied to the system either in the form of positivepressure to the sample well and the background fluid channels, ornegative pressure to the droplet outlet channel. In either case,sample-containing fluid is transported to droplet generation region 1124through sample fluid channel 1118, and background fluid is transportedto droplet generation region 1124 through background fluid channel 1112.Sample-containing droplets suspended in the background fluid are thenformed in the droplet generation region, from which they are transportedthrough droplet outlet channel 1114 to a droplet outlet 1126.

VI. Single Hole Mode Examples

This section describes examples of single hole mode droplet generationsystems, which are characterized by the fact that a sample fluid channeland a droplet outlet channel are formed by creating a single channelaperture through successive layers of material. This automaticallyresults in substantially perfect alignment of the sample fluid channeland the droplet outlet channel.

FIGS. 43-44 depict a first example of a single hole mode dropletgeneration system, generally indicated at 1150. Droplet generationsystem 1150 includes a substrate or droplet generator plate 1152, uponwhich are disposed a sample well 1154 and a background fluid well 1156.The terms “substrate” and “droplet generator plate” may be usedinterchangeably in the present teachings. The sample well and thebackground fluid well may be integrally formed with the substrate, forexample by injection molding, or in some cases they may be formedseparately and then attached to the substrate. Furthermore, substrate1152 may be substantially planar, as depicted in FIGS. 33-34, or it mayhave any other desired shape, such as a slightly curved cylindrical orspherical shape. Similarly, all of the other substrates described hereinas “substantially planar” may take other alternative forms according tothe present teachings.

As best seen in FIG. 44, substrate 1152 includes three layers of stackedmaterial 1158, 1160, 1162. These material layers may be bonded togetherby any suitable method, such as solvent bonding, gluing, or heatsealing, among others. Middle layer 1160 includes a central aperture1164, which in this example has an oval shape, but which in general canbe given any desired two-dimensional shape, or which may take the formof a substantially linear or non-linear channel. The key feature ofcentral aperture 1164 is that it extends between a region underneathsample well 1154 and a region underneath background fluid well 1156.

Background fluid well 1156 is configured to have an aperture 1166extending completely through upper material layer 1158, so thatbackground fluid well 1156 will automatically be fluidically connectedto central aperture 1164 when material layers 1158, 1160, and 1162 arestacked together. Sample well 1154 is configured to have an aperture1168 extending partially, but not completely, through upper materiallayer 1158. A channel 1170 is formed below aperture 1168, to fluidicallyinterconnect sample well 1154 and central aperture 1164. Channel 1170may be formed in a single operation, such as a drilling operation, aftermaterial layers 1158, 1160, and 1162 are assembled together. Thus,channel 1170 defines a sample channel 1170 a and a droplet channel 1170b, which will necessarily be in substantially perfect alignment witheach other. Alternatively, channel 1170 a may be formed in a separateoperation and then aligned to droplet channel 1170 b during assembly.

Central aperture 1164 defines a background fluid channel that intersectswith sample channel 1170 a and droplet channel 1170 b, to define adroplet generation region generally indicated at 1172. To create anemulsion of sample-containing droplets with system 1150,sample-containing fluid is placed in sample well 1154, and backgroundfluid is placed in background fluid well 1156. Positive pressure isapplied to the upper portions of the sample well and the backgroundfluid well, and/or negative pressure is applied to a droplet outletregion 1174 of the system. Background fluid is then transported throughthe background fluid channel defined by central aperture 1164, andsample-containing fluid is transported through sample channel 1170 a.These fluids intersect at droplet generation region 1172, to formsample-containing droplets suspended in the background fluid accordingto previously described principles. The resulting emulsion istransported through droplet channel 1170 b to droplet outlet region1174, where it may be collected and/or further transported as desired.

FIGS. 45-47 depict another example of a single hole mode dropletgeneration system, generally indicated at 1200. System 1200 is similarin some respects to system 1150. Specifically, system 1200 includes asubstrate 1202, upon which are disposed a sample well 1204 and abackground fluid well 1206. As in the case of system 1150, the samplewell and the background fluid well of system 1250 may or may not beintegrally formed with the substrate, and the substrate of system 1250may or may not be substantially planar.

In a slight distinction from system 1150, substrate 1202 of system 1200includes two primary layers of stacked material 1208, 1210, rather thanthree layers. These material layers again may be bonded together by anysuitable method. One of layers 1208, 1210, which in this example isupper layer 1208, includes a pair of circular depressions, 1212 a, 1212b, connected by a background fluid channel 1214. Channel 1214 is shownwith a two-dimensional rectangular shape, but can take any desired form,including a linear or non-linear elongate, substantially one-dimensionalchannel.

Background fluid well 1206 has an aperture 1216 extending through uppermaterial layer 1208, to fluidically connect background fluid well 1206with circular depression 1212 a and thus with background fluid channel1214. Similarly, sample well 1204 has an aperture 1218 extending throughupper material layer 1208. Furthermore, a complementary droplet outletchannel 1219 extends through lower material layer 1210. Sandwichedbetween material layers 1208 and 1210, and disposed within circulardepression 1212 a, is a disk stack generally indicated at 1220. Diskstack 1220 includes three disks 1220 a, 1220 b, 1220 c, stacked togetherand connected by any suitable method such as fusion welding or gluing.

When system 1200 is assembled, disk stack 1220 defines a dropletgeneration region as follows. Upper disk 1220 a includes a sample inlethole 1222 configured to transport sample-containing fluid from aperture1218 a through disk 1220 a. Middle disk 1220 b includes a backgroundfluid inlet portion 1224 configured to fluidically interconnect withbackground fluid channel 1214, and a droplet generation region 1226where sample-containing fluid emitted by sample inlet hole 1222intersects with background fluid transported through background fluidinlet 1224, to form sample-containing droplets suspended in backgroundfluid. Lower disk 1220 c includes a droplet outlet hole 1228, which isaligned with sample inlet hole 1222.

In some cases, sample inlet hole 1222 and droplet outlet hole 1228 maybe formed in a single operation such as by drilling the holes after diskstack 1220 is assembled, in which case the holes will have the same sizeand will automatically be substantially perfectly aligned. In othercases, however, it may be desirable to give the sample inlet hole andthe droplet outlet hole different diameters and/or geometries, forexample to control the rate of droplet formation by system 1200. Inthese cases, sample inlet hole 1222 and droplet outlet hole 1228 may beformed separately, before disk stack 1220 is assembled, and then alignedwith each other prior to assembly of the disk stack.

FIGS. 48-50 depict stylized sectional views of additional examples thatmay be characterized as single hole mode droplet generations systems. Asdescribed in more detail below, each of these systems includes aplurality of wells and channels that may be integrally formed as asingle component or as a pair of components that may be quickly andeasily joined together, with a sample fluid channel and a droplet outletchannel that may be formed in a single operation.

FIG. 48 is a stylized sectional view of a first example of the type ofsystem described in the previous paragraph. Specifically, FIG. 48depicts a droplet generation system, generally indicated at 1250, formedfrom two sections of material 1252 and 1254. Each of sections 1252, 1254may be injection molded, and configured to snap or otherwise fittogether in desired alignment. Alternatively, a similar exemplary systemmay be formed from a single piece of material, for example by injectionmolding, and then suitably processed to become a functional dropletgeneration system.

In any case, assembled system 1250 includes a sample well 1256, abackground fluid well 1258, and a droplet well 1260. As FIG. 48 shows,sample well 1256 is formed in material section 1252, whereas backgroundfluid well 1258 and droplet well 1260 are partially formed by each ofsections 1252 and 1254. Material section 1252 also defines asample-containing fluid channel 1262, which may be formed in section1252, for example, by drilling or laser scribing. If materials sections1252 and 1254 are joined together before fluid channel 1262 is formed,the same formation operation also may be used to form a first dropletchannel segment 1264 a, if that channel is not formed by the naturalinterface of sections 1252 and 1254.

Integration of sections 1252 and 1254 also results in the formation of abackground fluid outlet aperture 1266, a background fluid channel 1268,a droplet generation region generally indicated at 1270, a seconddroplet channel segment 1264 b, and a droplet outlet aperture 1272. Allof the described channels collectively form an integrated network ofchannels configured to fluidically interconnect the sample well, thebackground fluid well, and the droplet well, and to define dropletgeneration region 1270. Accordingly, when positive pressure is appliedto sample well 1256 and background fluid well 1258, and/or negativepressure is applied to droplet well 1260, sample-containing fluid andbackground fluid will each travel to droplet generation region 1270,where sample-containing droplets suspended in background fluid will begenerated. The resulting emulsion will then travel to, and be collectedin, droplet well 1260.

FIG. 49 shows a stylized sectional view of another example that can becharacterized as a single hole droplet generation system, generallyindicated at 1300. System 1300 is similar in many respects to system1250. As in the previous example, system 1300 includes two materialsections 1302 and 1304, which collectively define a sample well 1306, abackground fluid well 1308, a droplet well 1310, a sample-containingfluid channel 1312, a droplet channel 1314, a background fluid outletaperture 1316, a background fluid channel 1318, and a droplet generationregion generally indicated at 1320. In system 1300, however, dropletwell 1310 is disposed under the sample well and the background fluidwell (in the orientation of FIG. 49), and therefore may be characterizedas a catch well. Accordingly, system 1300 may be operated in conjunctionwith a centrifuge, to cause fluid transfer and droplet generation by theinertial forces associated with spinning the system.

FIG. 50 shows a stylized sectional view of still another example thatcan be characterized as a single hole droplet generation system,generally indicated at 1350. System 1350 is similar in many respects tosystems 1250 and 1300. As in the previous examples, system 1350 includestwo material sections 1352 and 1354, which collectively define a samplewell 1356, a background fluid well 1358, a droplet well 1360, asample-containing fluid channel 1362, a droplet channel 1364, abackground fluid outlet aperture 1366, a background fluid channel 1368,and a droplet generation region generally indicated at 1370.

System 1350, however, is configured so that sample-containing fluid andbackground fluid are respectively placed in sample well 1356 andbackground fluid well 1358 while the system is inverted relative to theorientation depicted in FIG. 50, and before material section 1354 isintegrated with section 1352. After material section 1354 is positionedto cover the sample well and the background fluid well, system 1350 thenmay be inverted to the orientation of FIG. 50, at which pointcompressing material sections 1352 and 1354 together will cause pressurewithin sample well 1356 and background fluid well 1358, and thus causedroplets to be generated and transported into droplet well 1360.

VII. Exemplary Methods of Operation

This section describes exemplary methods of operating droplet generationsystems, including at least some of the systems described above,according to aspects of the present teachings; see FIGS. 51-52.

FIG. 51 is a flowchart depicting an exemplary method, generallyindicated at 1400, of generating sample-containing droplets suspended ina background fluid according to aspects of the present teachings. Method1400 may be generally suitable for use with various droplet generationsystems described according to the present teachings, at least includingany of the systems shown in FIGS. 8-24 and described in the accompanyingtext above.

At step 1402, sample-containing fluid is transported into a sample wellattached to a substrate. At step 1404, background fluid is transportedinto a background fluid well attached to the substrate. At step 1406,sample-containing fluid is transported through a first channel formed inthe substrate, from the sample well to a droplet generation region. Atstep 1408, background fluid is transported through a second channelformed in the substrate, from the background fluid well to the dropletgeneration region. At step 1410, sample-containing droplets suspended inthe background fluid are generated at the droplet generation region. Atstep 1412, the sample-containing droplets are transported through athird channel formed in the substrate, from the droplet generationregion to a droplet outlet region attached to the substrate.

Method 1400 may include more detailed steps than the basic stepsdescribed so far. For example, transporting the sample-containing fluidthrough the first channel may include transporting the sample-containingfluid through an air trap region configured to prevent inadvertenttransport of the sample-containing fluid to the droplet generationregion. In addition, transporting background fluid through the secondchannel may include transporting the background fluid through twobackground fluid sub-channels that intersect the first channel from twodifferent directions to form a cross-shaped intersection region with thefirst channel and the third channel. Furthermore, generatingsample-containing droplets may include generating droplets havingvolumes in the range of 0.1 nanoliters to 10 nanoliters. Any otherdetails consistent with the disclosed droplet generation systems may beused in the steps of method 1400.

Aside from more details in the steps of method 1400, various additionalsteps may be performed. For example, method 1400 may include, asgenerally indicated at step 1409, applying negative pressure to thedroplet well and/or applying positive pressure to one or more of thesample well and the background fluid well, to cause transport of thefluids through the various channels and thus to cause dropletgeneration. As has been previously described, pressure may be applied byany suitable means, including at least pressure-controlled pumping,vacuum-controlled pumping, centrifugation, gravity-driven flow, andpositive displacement pumping.

FIG. 52 is a flow chart depicting another method, generally indicated at1450, for generating sample-containing droplets suspended in abackground fluid according to aspects of the present teachings. Asdescribed below, method 1450 includes the step of integrating at leasttwo components of a droplet generation system with each other, and thusmay be suitable for use with any of the systems described previouslythat include two or more separate components. At step 1452,sample-containing fluid is transported into a sample well. At step 1454,background fluid is transported into a background fluid well.

At step 1456, at least one of the sample well or the background fluidwell is integrated with a droplet generator housing, which may in somecases take the form of a substrate. At step 1458, sample-containingfluid is transported through a first channel formed in the housing, fromthe sample well to a droplet generation region. At step 1460, backgroundfluid is transported through a second channel formed in the housing,from the background fluid well to the droplet generation region. At step1462, sample-containing droplets suspended in the background fluid aregenerated at the droplet generation region. At step 1464, thesample-containing droplets are transported through a third channelformed in the substrate, from the droplet generation region to a dropletoutlet region attached to the substrate.

As in the case of method 1400, method 1450 may include more detailedsteps than the basic steps described above. For example, transportingthe sample-containing fluid through the first channel may includetransporting the sample-containing fluid through an air trap regionconfigured to prevent inadvertent transport of the sample-containingfluid to the droplet generation region, transporting background fluidthrough the second channel may include transporting the background fluidthrough two background fluid sub-channels that intersect the firstchannel from two different directions to form a cross-shapedintersection region with the first channel and the third channel, andgenerating sample-containing droplets may include generating dropletshaving volumes in the range of 0.1 nanoliters to 10 nanoliters. Anyother details consistent with the disclosed droplet generation systemsmay be used in the steps of method 1450.

Also as in the case of method 1400, various additional steps of method1450 may be performed. For example, as generally indicated at step 1461,method 1450 may include applying negative pressure to the droplet welland/or applying positive pressure to one or more of the sample well andthe background fluid well, to cause transport of the fluids through thevarious channels and thus to cause droplet generation. As has beenpreviously described, pressure may be applied by any suitable means,including at least pressure-controlled pumping, vacuum-controlledpumping, centrifugation, gravity-driven flow, and positive displacementpumping.

VIII. Exemplary Numbered Paragraphs

This section describes additional aspects and features of dropletgeneration for droplet-based assays, presented without limitation as aseries of numbered paragraphs.

Prototype (Two-Piece) Planar Mode

1. A system for forming a plurality of sample-containing dropletssuspended in a background fluid, comprising (A) a substrate having a topsurface and a bottom surface; (B) a sample port formed in the topsurface of the substrate; (C) a background fluid port formed in the topsurface of the substrate; (D) a droplet outlet port formed in the topsurface of the substrate; (E) a network of channels formed in the bottomsurface of the substrate and configured to fluidically interconnect thesample port, the background fluid port, and the droplet outlet port; (F)a droplet generation region defined by the network of channels andconfigured to generate sample-containing droplets suspended in thebackground fluid; and (G) a well vessel including a sample wellconfigured to make a substantially fluid tight connection with thesample port, a background fluid well configured to make a substantiallyfluid tight connection with the background fluid port, and a dropletoutlet well configured to make a substantially fluid tight connectionwith the droplet outlet port; wherein the droplet generation region isdefined by the intersection of at least a first channel, a secondchannel, and a third channel, and wherein the first channel isconfigured to transport sample-containing fluid from the sample port tothe droplet generation region, the second channel is configured totransport background fluid from the background fluid port to the dropletgeneration region, and the third channel is configured to transportsample-containing droplets from the droplet generation region to thedroplet outlet port.

Continuous Mode

2. A system for forming a plurality of sample-containing dropletssuspended in a background fluid, comprising (A) a sample well; (B) adroplet generator configured to receive sample-containing fluid from thesample well; (C) a droplet outlet region configured to receivesample-containing droplets from the droplet generator; and (D) a housingconfigured to selectively receive the droplet generator, the housingincluding a background fluid input channel configured to providebackground fluid to the droplet generator from a background fluidsource; wherein the droplet generator is configured to generatesample-containing droplets suspended in the background fluid, and todirect the droplets toward the droplet outlet region.

3. The system of paragraph 2, wherein at least one of the dropletgenerator and the droplet outlet region are integrally formed with thesample well.

Two-Part Mode

4. A system for forming a plurality of sample-containing dropletssuspended in a background fluid, comprising (A) a sample well; (B) asample channel configured to transport sample-containing fluid from thesample well to a droplet generation region; (C) a housing configured toselectively receive the sample channel; (D) a background fluid channelintegrally formed with the housing and configured to transportbackground fluid from a background fluid source to the dropletgeneration region; and (E) a droplet channel integrally formed with thehousing and configured to transport sample-containing droplets from thedroplet generation region to a droplet outlet; wherein the dropletgeneration region is disposed within the housing and is defined by aregion of intersection of the sample channel, the background fluidchannel, and the droplet channel.

5. The system of paragraph 4, wherein the sample channel is integrallyformed with the sample well.

Single-Hole Mode

6. A system for forming a plurality of sample-containing dropletssuspended in a background fluid, comprising (A) a droplet generatorplate; (B) a sample well attached to the droplet generator plate; (C) abackground fluid well attached to the droplet generator plate; (D) adroplet generation region formed within the droplet generator plate; (E)a background fluid channel formed within the droplet generator plate andconfigured to transport background fluid from the background fluid wellto the droplet generation region; (F) a sample channel configured totransport sample-containing fluid from the sample well to the dropletgeneration region; and (G) a droplet outlet channel configured totransport sample-containing droplets from the droplet generation regionto a droplet outlet formed in the droplet generator plate; wherein thesample inlet channel and the droplet outlet channel are integrallyformed from a pair of aligned apertures which are separated by thebackground fluid channel.

7. The system of paragraph 6, wherein the sample inlet channel and thedroplet outlet channel are each formed in the droplet generator plate.

8. The system of paragraph 7, wherein the sample inlet channel and thedroplet outlet channel are integrally formed by a single drillingoperation that passes through two rigidly attached planar surfaces ofthe droplet generator plate.

9. The system of paragraph 6, wherein the sample inlet channel and thedroplet outlet channel are each formed in an insertable dropletgenerator member configured to be disposed within the droplet generatorplate.

10. The system of paragraph 9, wherein the sample inlet channel and thedroplet outlet channel are integrally formed by a single drillingoperation that passes through two rigidly attached planar surfaces ofthe droplet generator member.

11. The system of paragraph 6, wherein at least one of the sample welland the background fluid well are integrally formed with the dropletgenerator plate.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

1-20. (canceled)
 21. A method of emulsion formation and modification,the method comprising: flowing an oil in an oil channel and a firstaqueous fluid in an aqueous channel, wherein the oil channel, theaqueous channel, and an emulsion channel intersect at a firstintersection; forming an emulsion including the first aqueous fluid atthe first intersection; flowing the emulsion in the emulsion channel toa second intersection; and modifying the emulsion at the secondintersection, wherein modifying comprises generating droplets includinga second aqueous fluid, wherein the modified emulsion comprises a firstpopulation of droplets including the first aqueous fluid and a secondpopulation of droplets including the second aqueous fluid, and whereinthe first population and the second population have differentcompositions.
 22. The method of claim 21, further comprising thermallycycling the first and second populations of droplets.
 23. The method ofclaim 21, further comprising amplifying a nucleic acid target in eachpopulation of droplets.
 24. The method of claim 21, further comprisingdetecting fluorescence from the first and second populations ofdroplets.
 25. The method of claim 21, further comprising placing thefirst aqueous fluid into a vessel; and removably connecting the vesselto the first intersection.
 26. The method of claim 25, furthercomprising driving the first aqueous fluid from the vessel to the firstintersection.
 27. The method of claim 21, wherein each of the first andsecond aqueous fluids includes a surfactant.
 28. The method of claim 21,wherein the oil includes a fluorocarbon oil.